Hyaluronan synthase gene and uses thereof

ABSTRACT

The present invention relates to a recombinant host cell that includes an expression construct having a hyaluronan synthase gene that encodes an enzymatically active hyaluronan synthase (HAS), and to the use of these recombinant cells that produce hyaluronan synthase and its hyaluronic acid product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/228,079, filedSep. 16, 2005; which is a continuation of U.S. Ser. No. 10/011,771,filed Dec. 11, 2001; which is a continuation of U.S. Ser. No.09/469,200, filed Dec. 21, 1999, now U.S. Pat. No. 6,833,264, issuedDec. 21, 2004, which is a continuation of U.S. Ser. No. 09/178,851,filed Oct. 26, 1998, now abandoned; which claims priority under 35U.S.C. § 119(e) of provisional application U.S. Ser. No. 60/064,435,filed Oct. 31, 1997; the entire contents of each of which is herebyexpressly incorporated herein by reference in their entirety. Saidapplication U.S. Ser. No. 10/011,771 is also a continuation-in-part ofU.S. Ser. No. 09/146,893, filed Sep. 3, 1998, now U.S. Pat. No.6,455,304, issued Sep. 24, 2002; which is a continuation of U.S. Ser.No. 08/270,581, filed Jul. 1, 1994, now abandoned; the entire contentsof each of which is hereby expressly incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The government owns certain rights in the present invention pursuant togrant number GM35978 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a nucleic acid segment having a codingregion segment encoding enzymatically active Streptococcus equisimilishyaluronate synthase (seHAS), and to the use of this nucleic acidsegment in the preparation of recombinant cells which producehyaluronate synthase and its hyaluronic acid product. Hyaluronate isalso known as hyaluronic acid or hyaluronan.

2. Brief Description of the Related Art

The incidence of streptococcal infections is a major health and economicproblem worldwide, particularly in developing countries. One reason forthis is due to the ability of Streptococcal bacteria to grow undetectedby the body's phagocytic cells, i.e., macrophages and polymorphonuclearcells (PMNs). These cells are responsible for recognizing and engulfingforeign microorganisms. One effective way the bacteria evadesurveillance is by coating themselves with polysaccharide capsules, suchas a hyaluronic acid (HA) capsule. The structure of HA is identical inboth prokaryotes and eukaryotes. Since HA is generally nonimmunogenic,the encapsulated bacteria do not elicit an immune response and are,therefore, not targeted for destruction. Moreover, the capsule exerts anantiphagocytic effect on PMNs in vitro and prevents attachment ofStreptococcus to macrophages. Precisely because of this, in Group A andGroup C Streptococci, the HA capsules are major virulence factors innatural and experimental infections. Group A Streptococcus areresponsible for numerous human diseases including pharyngitis, impetigo,deep tissue infections, rheumatic fever and a toxic shock-like syndrome.The Group C Streptococcus equisimilis is responsible for osteomyelitis,pharyngitis, brain abscesses, and pneumonia.

Structurally, HA is a high molecular weight linear polysaccharide ofrepeating disaccharide units consisting of N-acetylglucosamine (GlcNAc)and glucuronic acid (GlcA). The number of repeating disaccharides in anHA molecule can exceed 30,000, a M_(r)>10⁷. HA is the onlyglycosaminogylcan synthesized by both mammalian and bacterial cellsparticularly Groups A and C Streptococci and Type A Pasteurellamultocida. These strains make HA which is secreted into the medium aswell as HA capsules. The mechanism by which these bacteria synthesize HAis of broad interest medicinally since the production of the HA capsuleis a very efficient and clever way that Streptococci use to evadesurveillance by the immune system.

HA is synthesized by mammalian and bacterial cells by the enzymehyaluronate synthase which has been localized to the plasma membrane. Itis believed that the synthesis of HA in these organisms is a multi-stepprocess. Initiation involves binding of an initial precursor, UDP-GlcNAcor UDP-GlcA. This is followed by elongation which involves alternateaddition of the two sugars to the growing oligosaccharide chain. Thegrowing polymer is extruded across the plasma membrane region of thecell and into the extracellular space. Although the HA biosyntheticsystem was one of the first membrane heteropolysaccharide syntheticpathways studied, the mechanism of HA synthesis is still not wellunderstood. This may be because in vitro systems developed to date areinadequate in that de novo biosynthesis of HA has not been accomplished.

The direction of HA polymer growth is still a matter of disagreementamong those of ordinary skill in the art. Addition of themonosaccharides could be to the reducing or nonreducing end of thegrowing HA chain. Furthermore, questions remain concerning (i) whethernascent chains are linked covalently to a protein, to UDP or to a lipidintermediate, (ii) whether chains are initiated using a primer, and(iii) the mechanism by which the mature polymer is extruded through theplasma membrane of the Streptococcus. Understanding the mechanism of HAbiosynthesis may allow development of alternative strategies to controlStreptococcal and Pasteurella infections by interfering in the process.

HA has been identified in virtually every tissue in vertebrates and hasachieved widespread use in various clinical applications, most notablyand appropriately as an intra-articular matrix supplement and in eyesurgery. The scientific literature has also shown a transition from theoriginal perception that HA is primarily a passive structural componentin the matrix of a few connective tissues and in the capsule of certainstrains of bacteria to a recognition that this ubiquitous macromoleculeis dynamically involved in many biological processes: from modulatingcell migration and differentiation during embryogenesis to regulation ofextracellular matrix organization and metabolism to important roles inthe complex processes of metastasis, wound healing, and inflammation.Further, it is becoming clear that HA is highly metabolically active andthat cells focus much attention on the processes of its synthesis andcatabolism. For example, the half-life of HA in tissues ranges from 1 to3 weeks in cartilage to <1 day in epidermis.

It is now clear that a single protein utilizes both sugar substrates tosynthesize HA. The abbreviation HAS, for the HA synthase, has gainedwidespread support for designating this class of enzymes. Markovitz etal. successfully characterized the HAS activity from Streptococcuspyogenes and discovered the enzymes's membrane localization and itsrequirements for sugar nucleotide precursors and Mg²⁺. Prehm found thatelongating HA, made by B6 cells, was digested by hyaluronidase added tothe medium and proposed that HAS resides at the plasma membrane.Philipson and Schwartz also showed that HAS activity cofractionated withplasma membrane markers in mouse oligodendroglioma cells.

HAS assembles high M_(r) HA that is simultaneously extruded through themembrane into the extracellular space (or to make the cell capsule inthe case of bacteria) as glycosaminoglycan synthesis proceeds. This modeof biosynthesis is unique among macromolecules since nucleic acids,proteins, and lipids are synthesized in the nucleus, endoplasmicreticulum/Golgi, cytoplasm, or mitochondria. The extrusion of thegrowing chain into the extracellular space also allows for unconstrainedpolymer growth, thereby achieving the exceptionally large size of HA,whereas confinement of synthesis within a Golgi or post-Golgicompartment could limit the overall amount or length of the polymersformed. High concentrations of HA within a confined lumen could alsocreate a high viscosity environment that might be deleterious for otherorganelle functions.

Several studies attempted to solubilize, identify, and purify HAS fromstrains of Streptococci that make a capsular coat of HA as well as fromeukaryotic cells. Although the streptococcal and murineoligodendroglioma enzymes were successfully detergent-solubilized andstudied, efforts to purify an active HAS for further study or molecularcloning remained unsuccessful for decades. Prehm and Mausolf usedperiodate-oxidized UDP-GlcA or UDP-GlcNAc to affinity label a protein of−52 kDa in streptococcal membranes that co-purified with HAS. This ledto a report claiming that the Group C streptococcal HAS had been cloned,which was unfortunately erroneous. This study failed to demonstrateexpression of an active synthase and may have actually cloned a peptidetransporter. Triscott and van de Rijn used digitonin to solubilize HASfrom streptococcal membranes in an active form. Van de Rijn and Drakeselectively radiolabeled three streptococcal membrane proteins of 42,33, and 27 kDa with 5-azido-UDP-GlcA and suggested that the 33-kDaprotein was HAS. As shown later, however, HAS actually turned out to bethe 42-kDa protein.

Despite these efforts, progress in understanding the regulation andmechanisms of HA synthesis was essentially stalled, since there were nomolecular probes for HAS mRNA or HAS protein. A major breakthroughoccurred in 1993 when DeAngelis et al. reported the molecular cloningand characterization of the Group A streptococcal gene encoding theprotein HasA. This gene was known to be in part of an operon requiredfor bacterial HA synthesis, although the function of this protein, whichis now designated as spHAS (the S. pyogenes HAS), was unknown. spHAS wassubsequently proven to be responsible for HA elongation and was thefirst glycosaminoglycan synthase identified and cloned and thensuccessfully expressed. The S. pyogenes HA synthesis operon encodes twoother proteins. HasB is a UDP-glucose dehydrogenase, which is requiredto convert UDP-glucose to UDP-GlcA, one of the substrates for HAsynthesis. HasC is a UDP-glucose pyrophosphorylase, which is required toconvert glucose 1-phosphate and UTP to UDP-glucose. Co-transfection ofboth hasA and hasB genes into either acapsular Streptococcus strains orEnteroccus faecalis conferred them with the ability to synthesize HA andform a capsule. This provided the first strong evidence that HasA is anHA synthase.

The elusive HA synthase gene was finally cloned by a transposonmutagenesis approach, in which an acapsular mutant Group A strain wascreated containing a transposon interruption of the HA synthesis operon.Known sequences of the transposon allowed the region of the junctionwith streptococcal DNA to be identified and then cloned from wild-typecells. The encoded spHAS was 5-10% identical to a family of yeast chitinsynthases and 30% identical to the Xenopus laevis protein DG42(developmentally expressed during gastrulation), whose function wasunknown at the time. DeAngelis and Weigel expressed the activerecombinant spHAS in Escherichia coli and showed that this singlepurified gene product synthesizes high M_(r) HA when incubated in vitrowith UDP-GlcA and UDP-GlcNAc, thereby showing that bothglycosyltransferase activities required for HA synthesis are catalyzedby the same protein, as first proposed in 1959. This set the stage forthe almost simultaneous identification of eukaryotic HAS cDNAs in 1996by four laboratories revealing that HAS is a multigene family encodingdistinct isozymes. Two genes (HAS1 and HAS2) were quickly discovered inmammals (29-34), and a third gene HAS3 was later discovered. A secondstreptococcal seHAS or Streptococcus equisimilis hyaluronate synthase,has now been found and is the invention being claimed and disclosedherein.

As indicated, we have also identified the authentic HAS gene from GroupC Streptococcus equisimilis (seHAS); the seHAS protein has a high levelof identity (approximately 70 percent) to the spHAS enzyme. Thisidentity, however, is interesting because the seHAS gene does notcross-hybridize to the spHAS gene.

Membranes prepared from E. coli expressing recombinant seHAS synthesizeHA when both substrates are provided. The results confirm that theearlier report of Lansing et al. claiming to have cloned the Group C HASwas wrong. Unfortunately, several studies have employed antibody to thisuncharacterized 52-kDa streptococcal protein to investigate what wasbelieved to be eukaryotic HAS.

Itano and Kimata used expression cloning in a mutant mouse mammarycarcinoma cell line, unable to synthesize HA, to clone the firstputative mammalian HAS cDNA (mmHAS1). Subclones defective in HAsynthesis fell into three separate classes that were complementary forHA synthesis in somatic cell fusion experiments, suggesting that atleast three proteins are required. Two of these classes maintained someHA synthetic activity, whereas one showed none. The latter cell line wasused in transient transfection experiments with cDNA prepared from theparental cells to identify a single protein that restored HA syntheticactivity. Sequence analyses revealed a deduced primary structure for aprotein of ˜65 kDa with a predicted membrane topology similar to that ofspHAS. mmHAS1 is 30% identical to spHAS and 55% identical to DG42. Thesame month this report appeared, three other groups submitted papersdescribing cDNAs encoding what was initially thought to be the samemouse and human enzyme. However, through an extraordinary circumstance,each of the four laboratories had discovered a separate HAS isozyme inboth species.

Using a similar functional cloning approach to that of Itano and Kimata,Shyjan et al. identified the human homolog of HAS 1. A mesenteric lymphnode cDNA library was used to transfect murine mucosal T lymphocytesthat were then screened for their ability to adhere in a rosette assay.Adhesion of one transfectant was inhibited by antisera to CD44, a knowncell surface HA-binding protein, and was abrogated directly bypretreatment with hyaluronidase. Thus, rosetting by this transfectantrequired synthesis of HA. Cloning and sequencing of the responsible cDNAidentified hsHAS1. Itano and Kimata also reported a human HAS1 cDNAisolated from a fetal brain library. The hsHAS1 cDNAs reported by thetwo groups, however, differ in length; they encode a 578 or a 543 aminoacid protein. HAS activity has only been demonstrated for the longerform.

Based on the molecular identification of spHAS as an authentic HAsynthase and regions of near identity among DG42, spHAS, and NodC (aβ-GlcNAc transferase nodulation factor in Rhizobium), Spicer et al. useda degenerate RT-PCR approach to clone a mouse embryo cDNA encoding asecond distinct enzyme, which is designated mmHAS2. Transfection ofmmHAS2 cDNA into COS cells directed de novo production of an HA cellcoat detected by a particle exclusion assay, thereby providing strongevidence that the HAS2 protein can synthesize HA. Using a similarapproach, Watanabe and Yamaguchi screened a human fetal brain cDNAlibrary to identify hsHAS2. Fulop et al. independently used a similarstrategy to identify mmHAS2 in RNA isolated from ovarian cumulus cellsactively synthesizing HA, a critical process for normal cumulus oophorusexpansion in the pre-ovulatory follicle. Cumulus cell-oocyte complexeswere isolated from mice immediately after initiating an ovulatory cycle,before HA synthesis begins, and at later times when HA synthesis is justbeginning (3 h) or already apparent (4 h). RT-PCR showed that HAS2 mRNAwas absent initially but expressed at high levels 3-4 h later suggestingthat transcription of HAS2 regulates HA synthesis in this process. BothhsHAS2 are 552 amino acids in length and are 98% identical. mmHAS1 is583 amino acids long an 95% identical to hsHAS1, which is 578 aminoacids long.

Most recently Spicer et al. used a PCR approach to identify a third HASgene in mammals. The mmHAS3 protein is 554 amino acids long and 71, 56,and 28% identical, respectively, to mmHAS1, mmHAS2, DG42, and spHAS.Spicer et al. have also localized the three human and mouse genes tothree different chromosomes (HAS1 to hsChr 19/mmChr 17; HAS2 to hsChr8/mmChr 15; HAS3 to hsChr 16/mmChr 8). Localization of the three HASgenes on different chromosomes and the appearance of HA throughout thevertebrate class suggest that this gene family is ancient and thatisozymes appeared by duplication early in the evolution of vertebrates.The high identity (˜30%) between the bacterial and eukaryotic HASs alsosuggests that the two had a common ancestral gene. Perhaps primitivebacteria usurped the HAS gene from an early vertebrate ancestor beforethe eukaryotic gene products became larger and more complex.Alternatively, the bacteria could have obtained a larger vertebrate HASgene and deleted regulatory sequences nonessential for enzyme activity.

The discovery of X. laevis DG42 by Dawid and co-workers played asignificant role in these recent developments, even though this proteinwas not known to be an HA synthase. Nonetheless, that DG42 and spHASwere 30% identical was critical for designing oligonucleotides thatallowed identification of mammalian HAS2. Ironically, definitiveevidence that DG42 is a bona fide HA synthase was reported only afterthe discoveries of the Mammalian isozymes, when DeAngelis and Achyuthanexpressed the recombinant protein in yeast (an organism that cannotsynthesize HA) and showed that it synthesizes HA when isolated membranesare provided with the two substrates. Meyer and Kreil also showed thatlysates from cells transfected with cDNA for DG42 synthesize elevatedlevels of HA. Now that its function is known, DG42 can, therefore, bedesignated XIHAS.

There are common predicted structural features shared by all the HASproteins, including a large central domain and clusters of 2-3transmembrane or membrane-associated domains at both the amino andcarboxyl ends of the protein. The central domain, which comprises up to˜88% of the predicted intracellular HAS protein sequences, probablycontains the catalytic regions of the enzyme. This predicted centraldomain is 264 amino acids long in spHAS (63% of the total protein) and307-328 residues long in the eukaryotic HAS members (54-56% of the totalprotein). The exact number and orientation of membrane domains and thetopological organization of extracellular and intracellular loops havenot yet been experimentally determined for any HAS.

spHAS is a HAS family member that has been purified and partiallycharacterized. Initial studies using spHAS/alkaline phosphatase fusionproteins indicate that the N terminus, C terminus, and the large centraldomain of spHAS are, in fact, inside the cell. spHAS has 6 cysteines,whereas HAS1, HAS2, and HAS3 have 13, 14 and 14 Cys residues,respectively. Two of the 6 Cys residues in spHAS are conserved andidentical in HAS1 and HAS2. Only one conserved Cys residue is found atthe same position (Cys-225 in spHAS) in all the HAS family members. Thismay be an essential Cys whose modification by sulfhydryl poisonspartially inhibits enzyme activity. The possible presence of disulfidebonds or the identification of critical Cys residues needed for any ofthe multiple HAS functions noted below has not yet been elucidated forany members of the HAS family.

In addition to the proposed unique mode of synthesis at the plasmamembrane, the HAS enzyme family is highly unusual in the large number offunctions required for the overall polymerization of HA. At least sixdiscrete activities are present within the HAS enzyme: binding sites foreach of the two different sugar nucleotide precursors (UDP-GlcNAc andUDP-GlcA), two different glycosyltransferase activities, one or morebinding sites that anchor the growing HA polymer to the enzyme (perhapsrelated to a B-X₇-B motif), and a ratchet-like transfer reaction thatmoves the growing polymer one sugar at a time. This later activity islikely coincident with the stepwise advance of the polymer through themembrane. All of these functions, and perhaps others as yet unknown, arepresent in a relatively small protein ranging in size from 419 (spHAS)to 588 (xHAS) amino acids.

Although all the available evidence supports the conclusion that onlythe spHAS protein is required for HA biosynthesis in bacteria or invitro, it is possible that the larger eukaryotic HAS family members arepart of multicomponent complexes. Since the eukaryotic HAS proteins are˜40% larger than spHAS, their additional protein domains could beinvolved in more elaborate functions such as intracellular traffickingand localization, regulation of enzyme activity, and mediatinginteractions with other cellular components.

The unexpected finding that there are multiple vertebrate HAS genesencoding different synthases strongly supports the emerging consensusthat HA is an important regulator of cell behavior and not simply astructural component in tissues. Thus, in less than six months, thefield moved from one known, cloned HAS (spHAS) to recognition of amultigene family that promises rapid, numerous, and exciting futureadvances in our understanding of the synthesis and biology of HA.

For example, disclosed hereinafter are the sequences of the two HASgenes: from Pasteurella multocida; and (2) Paramecium bursaria chlorellavirus (PBCV-1). The presence of hyaluronan synthase in these two systemsand the purification and use of the hyaluronan synthase from these twodifferent systems indicates an ability to purify and isolate nucleicacid sequences encoding enzymatically active hyaluronan synthase in manydifferent prokaryotic and viral sources.

Group C Streptococcus equisimilis strain D181 synthesizes and secreteshyaluronic acid (HA). Investigators have used this strain and Group AStreptococcus pyogene strains, such as S43 and A111, to study thebiosynthesis of HA and to characterize the HA-synthesizing activity interms of its divalent cation requirement, precursor (UDP-GlcNAc andUDP-GlcA) utilization, and optimum pH.

Traditionally, HA has been prepared commercially by isolation fromeither rooster combs or extracellular media from Streptococcal cultures.One method which has been developed for preparing HA is through the useof cultures of HA-producing Streptococcal bacteria. U.S. Pat. No.4,517,295 describes such a procedure wherein HA-producing Streptococciare fermented under anaerobic conditions in a CO₂-enriched growthmedium. Under these conditions, HA is produced and can be extracted fromthe broth. It is generally felt that isolation of HA from rooster combsis laborious and difficult, since one starts with HA in a less purestate. The advantage of isolation from rooster combs is that the HAproduced is of higher molecular weight. However, preparation of HA bybacterial fermentation is easier, since the HA is of higher purity tostart with. Usually, however, the molecular weight of HA produced inthis way is smaller than that from rooster combs. Therefore, a techniquethat would allow the production of high molecular weight HA by bacterialfermentation would be an improvement over existing procedures.

High molecular weight HA has a wide variety of usefulapplications—ranging from cosmetics to eye surgery. Due to its potentialfor high viscosity and its high biocompatibility, HA finds particularapplication in eye surgery as a replacement for vitreous fluid. HA hasalso been used to treat racehorses for traumatic arthritis byintra-articular injections of HA, in shaving cream as a lubricant, andin a variety of cosmetic products due to its physiochemical propertiesof high viscosity and its ability to retain moisture for long periods oftime. In fact, in August 1997 the U.S. Food and Drug Agency approved theuse of high molecular weight HA in the treatment of severe arthritisthrough the injection of such high molecular weight HA directly into theaffected joints. In general, the higher molecular weight HA that isemployed the better. This is because HA solution viscosity increaseswith the average molecular weight of the individual HA polymer moleculesin the solution. Unfortunately, very high molecular weight HA, such asthat ranging up to 10⁷, has been difficult to obtain by currentlyavailable isolation procedures.

To address these or other difficulties, there is a need for new methodsand constructs that can be used to produce HA having one or moreimproved properties such as greater purity or ease of preparation. Inparticular, there is a need to develop methodology for the production oflarger amounts of relatively high molecular weight and relatively pureHA than is currently commercially available. There is yet another needto be able to develop methodology for the production of HA having amodified size distribution (HA_(Δsize)) as well as HA having a modifiedstructure (HA_(Δmod)).

The present invention addresses one or more shortcomings in the art.Using recombinant DNA technology, a purified nucleic acid segment havinga coding region encoding enzymatically active seHAS is disclosed andclaimed in conjunction, with methods to produce an enzymatically activeHA synthase, as well as methods for using the nucleic acid segment inthe preparation of recombinant cells which produce HAS and itshyaluronic acid product.

Thus, it is an object of the present invention to provide a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS.

It is a further object of the present invention to provide a recombinantvector which includes a purified nucleic acid segment having a codingregion encoding enzymatically active HAS.

It is still a further object of the present invention to provide arecombinant host cell transformed with a recombinant vector whichincludes a purified nucleic acid segment having a coding region encodingenzymatically active HAS.

It is yet another object of the present invention to provide a methodfor detecting a bacterial cell that expresses HAS.

It is another object of the present invention to provide a method forproducing high and/or low molecular weight hyaluronic acid from ahyaluronate synthase gene, such as seHAS, as well as methods forproducing HA having a modified size distribution and/or a modifiedstructure.

These and other objects of the present invention will become apparent inlight of the attached specification, claims, and drawings.

BRIEF SUMMARY OF THE INVENTION

The present invention involves the application of recombinant DNAtechnology to solving one or more problems in the art of hyaluronic acid(HA) preparation. These problems are addressed through the isolation anduse of a nucleic acid segment having a coding region encoding theenzymatically active Streptococcus equisimilis (seHAS) hyaluronatesynthase gene, a gene responsible for HA chain biosynthesis. The seHASgene was cloned from DNA of an appropriate microbial source andengineered into useful recombinant constructs for the preparation of HAand for the preparation of large quantities of the HAS enzyme itself.

The present invention encompasses a novel gene, seHAS. The expression ofthis gene correlates with virulence of Streptococcal Group A and Group Cstrains, by providing a means of escaping phagocytosis and immunesurveillance. The terms “hyaluronic acid synthase”, “hyaluronatesynthase”, “hyaluronan synthase” and “HA synthase”, are usedinterchangeably to describe an enzyme that polymerizes aglycosaminoglycan polysaccharide chain composed of alternatingglucuronic acid and N-acetylglucosamine sugars, β 1,3 and β 1,4 linked.The term “seHAS” describes the HAS enzyme derived from Streptococcusequisimilis.

The present invention concerns the isolation and characterization of ahyaluronate or hyaluronic acid synthase gene, cDNA, and gene product(HAS), as may be used for the polymerization of glucuronic acid andN-acetylglucosamine into the glycosaminoglycan hyaluronic acid. Thepresent invention identifies the seHAS locus and discloses the nucleicacid sequence which encodes for the enzymatically active seHAS gene fromStreptococcus equisimilis. The HAS gene also provides a new probe toassess the potential of bacterial specimens to produce hyaluronic acid.

Through the application of techniques and knowledge set forth herein,those of skill in the art will be able to obtain nucleic acid segmentsencoding the seHAS gene. As those of skill in the art will recognize, inlight of the present disclosure, these advantages provide significantutility in being able to control the expression of the seHAS gene andcontrol the nature of the seHAS gene product, the seHAS enzyme, that isproduced.

Accordingly, the invention is directed to the isolation of a purifiednucleic acid segment which has a coding region encoding enzymaticallyactive HAS, whether it be from prokaryotic or eukaryotic sources. Thisis possible because the enzyme, and indeed the gene, is one found inboth eukaryotes and some prokaryotes. Eukaryotes are also known toproduce HA and thus have HA synthase genes that can be employed inconnection with the invention.

HA synthase-encoding nucleic acid segments of the present invention aredefined as being isolated free of total chromosomal or genomic DNA suchthat they may be readily manipulated by recombinant DNA techniques.Accordingly, as used herein, the phrase “a purified nucleic acidsegment” refers to a DNA segment isolated free of unrelated chromosomalor genomic DNA and retained in a state rendering it useful for thepractice of recombinant techniques, such as DNA in the form of adiscrete isolated DNA fragment, or a vector (e.g., plasmid, phage orvirus) incorporating such a fragment.

A preferred embodiment of the present invention is a purified nucleicacid segment having a coding region encoding enzymatically active HAS.In particular, the purified nucleic acid segment encodes the seHAS ofSEQ ID NO:2 or the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:1.

Another embodiment of the present invention comprises a purified nucleicacid segment having a coding region encoding enzymatically active HASand the purified nucleic acid segment is capable of hybridizing to thenucleotide sequence of SEQ ID NO:1.

The present invention also comprises a natural or recombinant vectorconsisting of a plasmid, cosmid, phage, or virus vector. The recombinantvector may also comprise a purified nucleic acid segment having a codingregion encoding enzymatically active HAS.

In particular, the purified nucleic acid segment encodes the seHAS ofSEQ ID NO:2 or the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:1. If the recombinant vector is aplasmid, it may further comprise an expression vector. The expressionvector may also include a promoter operatively linked to theenzymatically active HAS coding region.

In another preferred embodiment, the present invention comprises arecombinant host cell such as a prokaryotic cell transformed with arecombinant vector. The recombinant vector includes a purified nucleicacid segment having a coding region encoding enzymatically active HAS.In particular, the purified nucleic acid segment encodes the seHAS ofSEQ ID NO:2 or the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:1.

The present invention also comprises a recombinant host cell, such as aneukaryotic cell transfected with a recombinant vector comprising apurified nucleic acid segment having a coding region encodingenzymatically active HAS. In particular, the purified nucleic acidsegment encodes the seHAS of SEQ ID NO:2 or the purified nucleic acidsegment comprises a nucleotide sequence in accordance with SEQ ID NO:1.The concept is to create a specifically modified seHAS gene that encodesan enzymatically active HAS capable of producing a hyaluronic acidpolymer having a modified structure or a modified size distribution.

The present invention further comprises a recombinant host cell which iselectroporated to introduce a recombinant vector into the recombinanthost cell. The recombinant vector may include a purified nucleic acidsegment having a coding region encoding enzymatically active HAS. Inparticular, the purified nucleic acid segment encodes the seHAS of SEQID NO:2 or the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:1. The enzymatically active HASmay also be capable of producing a hyaluronic acid polymer having amodified structure or a modified size distribution.

In yet another preferred embodiment, the present invention comprises arecombinant host cell which is transduced with a recombinant vectorwhich includes a purified nucleic acid segment having a coding regionencoding enzymatically active HAS. In particular, the purified nucleicacid segment encodes the seHAS of SEQ ID NO:2 or the purified nucleicacid segment comprises a nucleotide sequence in accordance with SEQ IDNO:1. The enzymatically active HAS is also capable of producing ahyaluronic acid polymer having a modified structure or a modified sizedistribution.

The present invention also comprises a purified composition, wherein thepurified composition comprises a polypeptide having a coding regionencoding enzymatically active HAS and further having an amino acidsequence in accordance with SEQ ID NO:2.

In another embodiment, the invention comprises a method for detecting aDNA species, comprising the steps of: (1) obtaining a DNA sample; (2)contacting the DNA sample with a purified nucleic acid segment inaccordance with SEQ ID NO:1; (3) hybridizing the DNA sample and thepurified nucleic acid segment thereby forming a hybridized complex; and(4) detecting the complex.

The present invention also comprises a method for detecting a bacterialcell that expresses mRNA encoding seHAS, comprising the steps of: (1)obtaining a bacterial cell sample; (2) contacting at least one nucleicacid from the bacterial cell sample with purified nucleic acid segmentin accordance with SEQ ID NO:1; (3) hybridizing the at least one nucleicacid and the purified nucleic acid segment thereby forming a hybridizedcomplex; and (4) detecting the hybridized complex, wherein the presenceof the hybridized complex is indicative of a bacterial strain thatexpresses mRNA encoding seHAS.

The present invention also comprises methods for detecting the presenceof either seHAS or spHAS in a cell. In particular, the method comprisesusing the oligonucleotides set forth in SEQ. ID NOS.: 3-8 as probes.These oligonucleotides would a allow a practitioner to search and detectthe presence of seHAS or spHAS in a cell.

The present invention further comprises a method for producinghyaluronic acid, comprising the steps of: (1) introducing a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS into a host organism, wherein the host organism containsnucleic acid segments encoding enzymes which produce UDP-GlcNAc andUDP-GlcA; (2) growing the host organism in a medium to secretehyaluronic acid; and (3) recovering the secreted hyaluronic acid.

The method may also include the step of extracting the secretedhyaluronic acid from the medium as well as the step of purifying theextracted hyaluronic acid. Furthermore, the host organism may secrete astructurally modified hyaluronic acid or a size modified hyaluronicacid.

The present invention further comprises a pharmaceutical compositioncomprising a preselected pharmaceutical drug and an effective amount ofhyaluronic acid produced by a recombinant HAS. The pharmaceuticalcomposition may have a hyaluronic acid having a modified molecularweight pharmaceutical composition capable of evading an immune response.The modified molecular weight may also produce a pharmaceuticalcomposition capable of targeting a specific tissue or cell type withinthe patient having an affinity for the modified molecular weightpharmaceutical composition.

The present invention also comprises a purified and isolated nucleicacid sequence encoding enzymatically active seHAS, where the nucleicacid sequence is (a) the nucleic acid sequence in accordance with SEQ IDNO:1; (b) complementary nucleic acid sequences to the nucleic acidsequence in accordance with SEQ ID NO:1; (c) nucleic acid sequenceswhich will hybridize to the nucleic acid in accordance with SEQ ID NO:1;and (d) nucleic acid sequences which will hybridize to the complementarynucleic acid sequences of SEQ ID NO:1.

The present invention further comprises a purified and isolated nucleicacid segment consisting essentially of a nucleic acid segment encodingenzymatically active HAS.

The present invention also comprises an isolated nucleic acid segmentconsisting essentially of a nucleic acid segment encoding seHAS having anucleic acid segment sufficiently duplicative of the nucleic acidsegment in accordance of SEQ ID NO:1 to allow possession of thebiological property of encoding for an enzymatically active HAS. Thenucleic acid segment may also be a cDNA sequence.

The present invention also comprises a purified nucleic acid segmenthaving a coding region encoding enzymatically active HAS, wherein thepurified nucleic acid segment is capable of hybridizing to thenucleotide sequence in accordance with SEQ ID NO:1.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts that cross hybridization between seHAS and spHAS genesdoes not occur.

FIG. 2 figuratively depicts the relatedness of seHAS to the bacterialand eukaryotic HAS proteins.

FIG. 3 figuratively depicts evolutionary relationships among some of theknown hyaluronan synthases.

FIG. 4 depicts the HA size distribution produced by various engineeredStreptococcal HAS enzymes.

FIG. 5 figuratively depicts the overexpression of recombinant seHAS andspHAS in E. coli.

FIG. 6 depicts purification of Streptococcal HA synthase.

FIG. 7 depicts a gel filtration analysis of HA synthesized byrecombinant streptococcal HAS expressed in yeast membranes.

FIG. 8 is a Western blot analysis of recombinant seHAS using specificantibodies.

FIG. 9 is a kinetic analysis of the HA size distributions produced byrecombinant seHAS and spHAS.

FIG. 10 graphically depicts the hydropathy plots for seHAS and predictedmembrane associated regions.

FIG. 11 is a graphical model for the topologic organization of seHAS inthe membrane.

FIG. 12 is a demonstration of the synthesis of authentic HA by therecombinant seHAS.

FIG. 13 depicts the recognition of nucleic acid sequences encodingseHAS, encoding spHAS, or encoding both seHAS and spHAS using specificoligonucleotides and PCR.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

As used herein, the term “nucleic acid segment” and “DNA segment” areused interchangeably and refer to a DNA molecule which has been isolatedfree of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Hyaluronate Synthase (“HAS”) coding sequenceyet is isolated away from, or purified free from, unrelated genomic DNA,for example, total Streptococcus equisimilis or, for example, mammalianhost genomic DNA. Included within the term “DNA segment”, are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

Similarly, a DNA segment comprising an isolated or purified seHAS generefers to a DNA segment including HAS coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case seHAS, formsthe significant part of the coding region of the DNA segment, and thatthe DNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HAS gene from prokaryotes such as S. pyogenes, S. equisimilis, or P.multocida. One such advantage is that, typically, eukaryotic enzymes mayrequire significant post-translational modifications that can only beachieved in a eukaryotic host. This will tend to limit the applicabilityof any eukaryotic HA synthase gene that is obtained. Moreover, those ofordinary skill in the art will likely realize additional advantages interms of time and ease of genetic manipulation where a prokaryoticenzyme gene is sought to be employed. These additional advantagesinclude (a) the ease of isolation of a prokaryotic gene because of therelatively small size of the genome and, therefore, the reduced amountof screening of the corresponding genomic library and (b) the ease ofmanipulation because the overall size of the coding region of aprokaryotic gene is significantly smaller due to the absence of introns.Furthermore, if the product of the seHAS gene (i.e., the enzyme)requires posttranslational modifications, these would best be achievedin a similar prokaryotic cellular environment (host) from which the genewas derived.

Preferably, DNA sequences in accordance with the present invention willfurther include genetic control regions which allow the expression ofthe sequence in a selected recombinant host. Of course, the nature ofthe control region employed will generally vary depending on theparticular use (e.g., cloning host) envisioned.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode a seHASgene, that includes within its amino acid sequence an amino acidsequence in accordance with SEQ ID NO:2. Moreover, in other particularembodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anHAS gene or DNA, and in particular to an HAS gene or cDNA, correspondingto Streptococcus equisimilis HAS. For example, where the DNA segment orvector encodes a full length HAS protein, or is intended for use inexpressing the HAS protein, preferred sequences are those which areessentially as set forth in SEQ ID NO:2.

Nucleic acid segments having HA synthase activity may be isolated by themethods described herein. The term “a sequence essentially as set forthin SEQ ID NO:2” means that the sequence substantially corresponds to aportion of SEQ ID NO:2 and has relatively few amino acids which are notidentical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:2. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as agene having a sequence essentially as set forth in SEQ ID NO:2, and thatis associated with the ability of prokaryotes to produce HA or ahyaluronic acid coat.

For instance, the seHAS and spHAS coding sequences are approximately 70%identical and rich in the bases adenine (A) and thymine (T). SeHAS basecontent is A-26.71%, C-19.13%, G-20.81%, and T-33.33% (A/T=60%). WhereasspHAS is A-31.34%, C-16.42%, G-16.34%, and T-35.8% (A/T=67%). Those ofordinary skill in the art would be surprised that the seHAS codingsequence does not hybridize with the spHAS gene and vice versa, despitetheir being 70% identical. This unexpected inability to cross-hybridizecould be due to short interruptions of mismatched bases throughout theopen reading frames. The inability of spHAS and seHAS to cross-hybridizeis shown in FIG. 1. The longest stretch of identical nucleotides commonto both the seHAS and the spHAS coding sequences is only 20 nucleotides.In addition, the very A-T rich sequences will form less stablehybridization complexes than G-C rich sequences. Another possibleexplanation could be that there are several stretches of As or Ts inboth sequences that could hybridize in a misaligned and unstable manner.This would put the seHAS and spHAS gene sequences out of frame withrespect to each other, thereby decreasing the probability of productivehybridization.

Because of this unique phenomena of two genes encoding proteins whichare 70% identical not being capable of cross-hybridizing to one another,it is beneficial to think of the claimed nucleic acid segment in termsof its function; i.e., a nucleic acid segment which encodesenzymatically active hyaluronate synthase. One of ordinary skill in theart would appreciate that a nucleic acid segment encoding enzymaticallyactive hyaluronate synthase may contain conserved or semi-conservedsubstitutions to the sequences set forth in SEQ ID NOS: 1 and 2 and yetstill be within the scope of the invention.

In particular, the art is replete with examples of practitioners abilityto make structural changes to a nucleic acid segment (i.e., encodingconserved or semi-conserved amino acid substitutions) and still preserveits enzymatic or functional activity. See for example: (1) Risler et al.“Amino Acid Substitutions in Structurally Related Proteins. A PatternRecognition Approach.” J. Mol. Biol. 204:1019-1029 (1988) [“ . . .according to the observed exchangeability of amino acid side chains,only four groups could be delineated; (i) Ile and Val; (ii) Leu and Met,(iii) Lys, Arg, and Gin, and (iv) Tyr and Phe.”]; (2) Niefind et al.“Amino Acid Similarity Coefficients for Protein Modeling and SequenceAlignment Derived from Main-Chain Folding Anoles.” J. Mol. Biol.219:481-497 (1991) [similarity parameters allow amino acid substitutionsto be designed]; and (3) Overington et al. “Environment-Specific AminoAcid Substitution Tables: Tertiary Templates and Prediction of ProteinFolds,” Protein Science 1:216-226 (1992) [“Analysis of the pattern ofobserved substitutions as a function of local environment shows thatthere are distinct patterns . . . ” Compatible changes can be made.]

These references and countless others, indicate that one of ordinaryskill in the art, given a nucleic acid sequence, could makesubstitutions and changes to the nucleic acid sequence without changingits functionality. Also, a substituted nucleic acid segment may behighly identical and retain its enzymatic activity with regard to itsunadulterated parent, and yet still fail to hybridize thereto.

The invention discloses nucleic acid segments encoding enzymaticallyactive hyaluronate synthase—seHAS and spHAS. Although seHAS and spHASare 70% identical and both encode enzymatically active hyaluronatesynthase, they do not cross hybridize. Thus, one of ordinary skill inthe art would appreciate that substitutions can be made to the seHASnucleic acid segment listed in SEQ ID NO:1 without deviating outside thescope and claims of the present invention. Standardized and acceptedfunctionally equivalent amino acid substitutions are presented in TableI. TABLE I Conservative and Semi- Amino Acid Group ConservativeSubstitutions NonPolar R Groups Alanine, Valine, Leucine, Isoleucine,Proline, Methionine, Phenylalanine, Tryptophan Polar, but uncharged, RGroups Glycine, Serine, Threonine, Cysteine, Asparagine, GlutamineNegatively Charged R Groups Aspartic Acid, Glutamic Acid PositivelyCharged R Groups Lysine, Arginine, Histidine

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:2, further defined as a recombinant vector. As used herein, the term“recombinant vector” refers to a vector that has been modified tocontain a nucleic acid segment that encodes an HAS protein, or fragmentthereof. The recombinant vector may be further defined as an expressionvector comprising a promoter operatively linked to said HAS encodingnucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising an HAS gene. Thepreferred recombinant host cell may be a prokaryotic cell. In anotherembodiment, the recombinant host cell is a eukaryotic cell. As usedherein, the term “engineered” or “recombinant” cell is intended to referto a cell into which a recombinant gene, such as a gene encoding HAS,has been introduced. Therefore, engineered cells are distinguishablefrom naturally occurring cells which do not contain a recombinantlyintroduced gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinantly introduced genes willeither be in the form of a cDNA gene, a copy of a genomic gene, or willinclude genes positioned adjacent to a promoter not naturally associatedwith the particular introduced gene.

Where one desires to use a host other than Streptococcus, as may be usedto produce recombinant HA synthase, it may be advantageous to employ aprokaryotic system such as E. coli, B. subtilis, Lactococcus sp., oreven eukaryotic systems such as yeast or Chinese hamster ovary, Africangreen monkey kidney cells, VERO cells, or the like. Of course, wherethis is undertaken it will generally be desirable to bring the HAsynthase gene under the control of sequences which are functional in theselected alternative host. The appropriate DNA control sequences, aswell as their construction and use, are generally well known in the artas discussed in more detail hereinbelow.

In preferred embodiments, the HA synthase-encoding DNA segments furtherinclude DNA sequences, known in the art functionally as origins ofreplication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which HA synthase DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin bacterial hosts suitable for biotechnology applications. However, formore versatility of cloned DNA segments, it may be desirable toalternatively or even additionally employ origins recognized by otherhost systems whose use is contemplated (such as in a shuttle vector).

The isolation and use of other replication origins such as the SV40,polyoma or bovine papilloma virus origins, which may be employed forcloning or expression in a number of higher organisms, are well known tothose of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the HA synthase coding gene sequence together withan appropriate replication origin and under the control of selectedcontrol regions.

Thus, it will be appreciated by those of skill in the art that othermeans may be used to obtain the HAS gene or cDNA, in light of thepresent disclosure. For example, polymerase chain reaction or RT-PCRproduced DNA fragments may be obtained which contain full complements ofgenes or cDNAs from a number of sources, including other strains ofStreptococcus or from eukaryotic sources, such as cDNA libraries.Virtually any molecular cloning approach may be employed for thegeneration of DNA fragments in accordance with the present invention.Thus, the only limitation generally on the particular method employedfor DNA isolation is that the isolated nucleic acids should encode abiologically functional equivalent HA synthase.

Once the DNA has been isolated it is ligated together with a selectedvector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli areemployed.

Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coli,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of HA. These are benign and well studiedorganisms used in the production of certain foods and biotechnologyproducts. These are advantageous in that one can augment the Lactococcusor Bacillus strain's ability to synthesize HA through gene dosaging(i.e., providing extra copies of the HA synthase gene by amplification)and/or inclusion of additional genes to increase the availability of HAprecursors. The inherent ability of a bacterium to synthesize HA canalso be augmented through the formation of extra copies, oramplification, of the plasmid that carries the HA synthase gene. Thisamplification can account for up to a 10-fold increase in plasmid copynumber and, therefore, the HA synthase gene copy number.

Another procedure that would further augment HA synthase gene copynumber is the insertion of multiple copies of the gene into the plasmid.Another technique would include integrating the HAS gene intochromosomal DNA. This extra amplification would be especially feasible,since the bacterial HA synthase gene size is small. In some scenarios,the chromosomal DNA-ligated vector is employed to transfect the hostthat is selected for clonal screening purposes such as E. coli, throughthe use of a vector that is capable of expressing the inserted DNA inthe chosen host.

Where a eukaryotic source such as dermal or synovial fibroblasts orrooster comb cells is employed, one will desire to proceed initially bypreparing a cDNA library. This is carried out first by isolation of mRNAfrom the above cells, followed by preparation of double stranded cDNAusing an enzyme with reverse transcriptase activity and ligation withthe selected vector. Numerous possibilities are available and known inthe art for the preparation of the double stranded cDNA, and all suchtechniques are believed to be applicable. A preferred technique involvesreverse transcription. Once a population of double stranded cDNAs isobtained, a cDNA library is prepared in the selected host by acceptedtechniques, such as by ligation into the appropriate vector andamplification in the appropriate host. Due to the high number of clonesthat are obtained, and the relative ease of screening large numbers ofclones by the techniques set forth herein, one may desire to employphage expression vectors, such as λgt11, λgt12, λGem11, and/or λZAP forthe cloning and expression screening of cDNA clones.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table I, and also refers tocodons that encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ nucleic acid sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes. In particular, the amino acidsequence of the HAS gene in eukaryotes appears to be 40% larger thanthat found in prokaryotes.

Allowing for the degeneracy of the genetic code as well as conserved andsemi-conserved substitutions, sequences which have between about 40% andabout 80%; or more preferably, between about 80% and about 90%; or evenmore preferably, between about 90% and about 99%; of nucleotides whichare identical to the nucleotides of SEQ ID NO:1 will be sequences whichare “essentially as set forth in SEQ ID NO:1”. Sequences which areessentially the same as those set forth in SEQ ID NO:1 may also befunctionally defined as sequences which are capable of hybridizing to anucleic acid segment containing the complement of SEQ ID NO:1 understandard or less stringent hybridizing conditions. Suitable standardhybridization conditions will be well known to those of skill in the artand are clearly set forth herein.

The term “standard hybridization conditions” as used herein, is used todescribe those conditions under which substantially complementarynucleic acid segments will form standard Watson-Crick base-pairing. Anumber of factors are known that determine the specificity of binding orhybridization, such as pH, temperature, salt concentration, the presenceof agents, such as formamide and dimethyl sulfoxide, the length of thesegments that are hybridizing, and the like. When it is contemplatedthat shorter nucleic acid segments will be used for hybridization, forexample fragments between about 14 and about 100 nucleotides, salt andtemperature preferred conditions for hybridization will include1.2-1.8×HPB at 40-50° C.

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1. Nucleic acid sequences which are “complementary” arethose which are capable of base-pairing according to the standardWatson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. It is therefore contemplated thata nucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NOS:1and 2. Recombinant vectors and isolated DNA segments may thereforevariously include the HAS coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides which neverthelessinclude HAS-coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

For instance, we have found, characterized, and purified hyaluronatesynthase in two other systems: (a) the gram-negative bacteriaPasteurella multocida (SEQ ID NO:9); and (2) chlorella virus PBCV-1 (SEQID NOS:7 and 8). The presence of hyaluronan synthase in these twosystems and our ability to purify and use the hyaluronan synthase fromthese two different systems indicates our ability to purify and isolatenucleic acid sequences encoding enzymatically active hyaluronansynthase.

The capsule of Carter Type A P. multocida (SEQ ID NO:9) was longsuspected of containing hyaluronic acid-HA. Characterization of the HAsynthase of P. multocida led to interesting enzymological differencesbetween it and the seHAS and spHAS proteins.

P. multocida cells produce a readily visible extracellular HA capsule,and since the two streptococcal HASs are membrane proteins, membranepreparations of the fowl cholera pathogen were tested. In early trials,crude membrane fractions derived from ultrasonication alone possessedvery low levels of UDP-GlcNAc-dependent UDP-[¹⁴C]GlcA incorporation intoHA[˜0.2 pmol of GlcA transfer (μg of proteins)⁻¹h⁻¹] when assayed underconditions similar to those for measuring streptococcal HAS activity.The enzyme from E. coli with the recombinant hasA plasmid was alsorecalcitrant to isolation at first. These results were in contrast tothe easily detectable amounts obtained from Streptococcus by similarmethods.

An alternative preparation protocol using ice-cold lysozyme treatment inthe presence of protease inhibitors in conjunction with ultrasonicationallowed the substantial recovery of HAS activity from both species ofGram-negative bacteria. Specific activities for HAS of 5-10 pmol of GlcAtransferred (μg of protein)⁻¹h⁻¹ were routinely obtained for crudemembranes of wild-type P. multocida with the new method. In the absenceof UDP-GlcNAc, virtually no radioactivity (<1% of identical assay withboth sugar precursors) from UDP-[¹⁴C]GlcA was incorporated into highermolecular weight material. Membranes prepared from the acapsular mutant,TnA, possessed no detectable HAS activity when supplemented with bothsugar nucleotide precursors (data not shown). Gel-filtration analysisusing a Sephacryl S-200 column indicates that the molecular mass of themajority of the ¹⁴C-labeled product synthesized in vitro is ≧8×10⁴ Dasince the material elutes in the void volumes, such a value correspondsto a HA molecule composed of at least 400 monomers. This product issensitive to Streptomyces hyaluronidase digestion but resistant toprotease treatment.

The parameters of the HAS assay were varied to maximize incorporation ofUDP-sugars into polysaccharide by P. multocida membranes. StreptococcalspHAS requires Mg²⁺ and therefore this metal ion was included in theinitial assays of P. multocida membranes. The P. multocida HAS (pmHAS)was relatively active from pH 6.5 to 8.6 in Tris-type buffers with anoptimum at pH 7. The HAS activity was linear with respect to theincubation time at neutral pH for at least 1 h. The pmHAS was apparentlyless active at higher ionic strengths because the addition of 100 mMNaCl to the reaction containing 50 mM Tris, pH 7, and 20 mM MgCl₂reduced sugar incorporation by 50%.

The metal ion specificity of the pmHAS was assessed at pH 7. Undermetal-free conditions in the presence of EDTA, no incorporation ofradiolabeled precursor into polysaccharide was detectable (<0.5% ofmaximal signal). Mn²⁺ gave the highest incorporation rates at the lowestion concentrations for the tested metals (Mg, Mn, Co, Cu, and Ni). Mg²⁺gave about 50% of the Mn²⁺ stimulation but at 10-fold higherconcentrations. Co²⁺ or Ni²⁺ at 10 mM supported lower levels of activity(20% or 9%, respectively, of 1 mM Mn²⁺ assays), but membranes suppliedwith 10 mM Cu²⁺ were inactive. Indeed, mixing 10 mM Cu²⁺ and 20 mM²⁺Mg²⁺ with the membrane preparation resulted in almost no incorporationof label into polysaccharide (<0.8% of Mg only value).

Initial characterization of the pmHAS was performed in the presence ofMg²⁺. The binding affinity of the enzyme for its sugar nucleotideprecursors was assessed by measuring the apparent K_(M) value.Incorporation of [¹⁴C]GlcA or [³H]GlcNAc into polysaccharide wasmonitored at varied concentrations of UDP-GlcNAc or UDP-GlcA,respectively. In Mg²⁺-containing buffers, the apparent K_(M) values of˜20 μM for UDP-GlcA and ˜75 μM for UDP-GlcNAc were determined utilizingHanes-Woolf plots ([S]/v versus [S]) of the titration data. The V_(max)values for both sugars were the same because the slopes, correspondingto 1/V_(max), of the Hanes-Woolf plots were equivalent. In comparison toresults from assays with Mg²⁺, the KM value for UDP-GlcNAc was increasedby about 25-50% to ˜105 μM and the V_(max) increased by a factor of2-3-fold in the presence of Mn²⁺.

The HA synthase enzymes from either P. multocida, S. equisimilis, or S.pyogenes utilizes UDP-sugars, but they possess somewhat differentkinetic optima with respect to pH and metal ion dependence and KMvalues. The enzymes are most active at pH 7; however, the pmHASreportedly displays more activity at slightly acidic pH and isrelatively inactive above pH 7.4. The pmHAS utilizes Mn²⁺ moreefficiently than Mg²⁺ under the in vitro assay conditions, but theidentity of the physiological metal cofactor in the bacterial cell isunknown. In comparison, in previous studies with the streptococcalenzyme, Mg²⁺ was much better than Mn²⁺ but the albeit smaller effect ofMn²⁺ was maximal at −10-fold lower concentrations than the optimal Mg²⁺concentration. The pmHAS apparently binds the UDP-sugars more tightlythan spHAS. The measured K_(M) values for the pmHAS in crude membranesare about 2-3-fold lower for each substrate than those obtained from theHAS found in streptococcal membranes: 50 or 39 μM for UDP-GlcA and 500or 150 μM for UDP-GlcNAc, respectively.

By kinetic analyses, the V_(max) of the pmHAS was 2-3-fold higher in thepresence of Mn²⁺ than Mg²⁺, but the UDP-GlcNAc K_(M) value was increasedslightly in assays with the former ion. This observation of apparentlowered affinity suggests that the increased polymerization rate was notdue to better binding of the Mn²⁺ ion/sugar nucleotide complex to theenzyme active site(s). Therefore, it is possible that Mn²⁺ enhances someother reaction step, alters another site/structure of the enzyme, ormodifies the phospholipid membrane environment. The gene sequence andthe protein sequence of pmHAS are shown in SEQ ID NO:9.

Chlorella virus PBCV-1 encodes a functional glycosyltransferase that cansynthesize a polysaccharide, hyaluronan [hyaluronic acid, HA]. Thisfinding is contrary to the general observation that viruses either: (a)utilize host cell glycosyltransferases to create new carbohydratestructures, or (b) accumulate host cell glycoconjugates during virionmaturation. Furthermore, HA has been generally regarded as restricted toanimals and a few of their virulent bacterial pathogens. Though manyplant carbohydrates have been characterized, neither HA nor a relatedanalog has previously been detected in cells of plants or protists.

The vertebrate HAS enzymes (DG42, HAS1, HAS2, HAS3) and streptococcalHasA enzymes (spHAS and seHAS) have several regions of sequencesimilarity. While sequencing the double-stranded DNA genome of virusPBCV-1 [Paramecium bursaria chlorella virus], an ORF [open readingframe], A98R (Accession #442580), encoding a 567 residue protein with 28to 33% amino acid identity to the various HASs was discovered. Thisprotein is designated cvHAS (chlorella virus HA synthase). The genesequence encoding PBCV-1 and its protein sequence are shown in SEQ IDNOS:7 and 8.

PBCV-1 is the prototype of a family (Phycodnarviridae) of large (175-190nm diameter) polyhedral, plaque-forming viruses that replicate incertain unicellular, eukaryotic chlorella-like green algae. PBCV-1virions contain at least 50 different proteins and a lipid componentlocated inside the outer glycoprotein capsid. The PBCV-1 genome is alinear, nonpermuted 330-kb dsDNA molecule with covalently closed hairpinends.

Based on its deduced amino acid sequence, the A98R gene product shouldbe an integral membrane protein. To test this hypothesis, recombinantA98R was produced in Escherichia coli and the membrane fraction wasassayed for HAS activity. UDP-GlcA and UDP-GlcNAc were incorporated intothe polysaccharide by the membrane fraction derived from cellscontaining the A98R gene on a plasmid, pCVHAS, (average specificactivity 2.5 pmoles GlcA transfer/μg protein/min) but not by samplesfrom control cells (<0.001 pmoles GlcA transfer/μg protein/min). Noactivity was detected in the soluble fraction of cells transformed withpCVHAS. UDP-GlcA and UDP-GlcNAc were simultaneously required forpolymerization. The activity was optimal in Hepes buffer at pH 7.2 inthe presence of 10 mM MnCl₂, whereas no activity was detected if themetal ion was omitted. Mg²⁺ and Co²⁺ were ˜20% as effective as Mn²⁺ atsimilar concentrations. The pmHAS has a similar metal requirement, butother HASs prefer Mg²⁺.

The recombinant A98R enzyme synthesized a polysaccharide with an averagemolecular weight of 3-6×10⁶ Da which is smaller than that of the HAsynthesized by recombinant spHAS or DG42 xlHAS in vitro (˜10⁷ Da and˜5-8×10⁶ Da, respectively; 13, 15). The polysaccharide was completelydegraded by Streptomyces hyaluroniticus HA lyase, an enzyme thatdepolymerizes HA, but not structurally related glycosaminoglycans suchas heparin and chondroitin.

PBCV-1 infected chlorella cells were examined for A98R gene expression.A ˜1,700-nucleotide A98R transcript appeared at ˜15 min post-infectionand disappeared by 60 min after infection indicating that A98R is anearly gene. Consequently, membrane fractions from uninfected and PBCV-1infected chlorella cells were assayed at 50 and 90 min post-infectionfor HAS activity. Infected cells, but not uninfected cells, hadactivity. Like the bacterially derived recombinant A98R enzyme,radiolabel incorporation from UDP-[¹⁴C]GlcA into polysaccharide dependedon both Mn²⁺ and UDP-GlcNAc. This radiolabeled produce was also degradedby HA lyase. Disrupted PBCV-1 virions had no HAS activity.

PBCV-1 infected chlorella cells were analyzed for HA polysaccharideusing a highly specific ¹²⁵I-labeled HA-binding protein. Extracts fromcells at 50 and 90 min post-infection contained substantial amounts ofHA, but not extracts from uninfected algae or disrupted PBCV-1 virions.The labeled HA-binding protein also interacted with intact infectedcells at 50 and 90 min post-infection, but not healthy cells. Therefore,a considerable portion of the newly synthesized HA polysaccharide wasimmobilized at the outer cell surface of the infected algae. Theextracellular HA does not play any obvious role in the interactionbetween the virus and its algal host because neither plaque size norplaque number was altered by including either testicular hyaluronidase(465 units/ml) or free HA polysaccharide (100 μg/ml) in the top agar ofthe PBCV-1 plaque assay.

The PBCV-1 genome also has additional genes that encode for an UDP-Glcdehydrogenase (UDP-Glc DH) and a glutamine:fructose-6-phosphateaminotransferase (GFAT). UDP-Glc DH converts UDP-Glc into UDP-GlcA, arequired precursor for HA biosynthesis. GFAT convertsfructose-6-phosphate into glucosamine-6-phosphate, an intermediate inthe UDP-GlcNAc metabolic pathway. Both of these PBCV-1 genes, like theA98R HAS, are expressed early in infection and encode enzymaticallyactive proteins. The presence of multiple enzymes in the HA biosynthesispathway indicates that HA production must serve an important function inthe life cycle of the chlorella viruses.

HA synthases of Streptococcus, vertebrates, and PBCV-1 possess manymotifs of 2 to 4 residues that occur in the same relative order. Theseconserved motifs probably reflect domains crucial for HA biosynthesis asshown in FIG. 2. The protein sequences of Group C seHAS, Group A spHAS,murine HAS1, HAS2, HAS3, and frog HAS are shown aligned in FIG. 2. Thealignment of FIG. 2 was accomplished using the DNASIS multiple alignmentprogram. Residues in seHAS identical in other known HAS family members(including human HAS1 and 2, not shown) are denoted by shading andasterisks. The amino acids indicated by dots are conserved in allmembers of the larger β-glycosyl transferase family. The diamond symbolindicates the highly conserved cysteine residue that may be critical forenzyme activity. The approximate mid-points of predicted membranedomains MD1 through MD7 are indicated with arrows. X1 indicates Xeopuslaevis, and MM denotes Mus musculis.

Regions of similarity between HASs and other enzymes that synthesizeβ-linked polysaccharides from UDP-sugar precursors are also beingdiscovered as more glycosyltransferases are sequenced. Examples includebacterial cellulose synthase, fungal and bacterial chitin synthases, andthe various HASs. The significance of these similar structural motifswill become more apparent as the three-dimensional structures ofglycosyltransferases accumulate.

FIG. 3 depicts the evolutionary relationships among the known hyaluronansynthase. The phylogenetic tree of FIG. 3 was generated by theHiggins-Sharp algorithm using the DNAsis multiple alignment program. Thecalculated matching percentages are indicated at each branch of thedendrogram.

The DNA segments of the present invention encompass biologicallyfunctional equivalent HAS proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HASprotein or to test HAS mutants in order to examine HA synthase activityat the molecular level.

Also, specific changes to the HAS coding sequence can result in theproduction of HA having a modified size distribution or structuralconfiguration. One of ordinary skill in the art would appreciate thatthe HAS coding sequence can be manipulated in a manner to produce analtered hyaluronate synthase which in turn is capable of producinghyaluronic acid having differing polymer sizes and/or functionalcapabilities. For example, the HAS coding sequence may be altered insuch a manner that the hyaluronate synthase has an altered sugarsubstrate specificity so that the hyaluronate synthase creates a newhyaluronic acid-like polymer incorporating a different structure such asa previously unincorporated sugar or sugar derivative. This newlyincorporated sugar could result in a modified hyaluronic acid havingdifferent functional properties, a hyaluronic acid having a smaller orlarger polymer size/molecular weight, or both. As will be appreciated byone of ordinary skill in the art given the HAS coding sequences, changesand/or substitutions can be made to the HAS coding sequence such thatthese desired property and/or size modifications can be accomplished.Table II lists sugar nucleotide specificity and magnesium ionrequirement of recombinant seHAS. TABLE II Sugar nucleotide specificityand Magnesium ion requirement of recombinant seHAS HA Synthesis* SecondSugar nucleotide UDP-[¹⁴C]GlcA UDP-[³H]GlcNAc present (μM) dpm (%) dpm(%) None 90 (2.1%)  8 (1.2%) UDP-GlcNAc (300) 4134 (100%)   — UDP-GlcA(120) — 635 (100%)  UDP-Glc (160) 81 (1.9%) 10 (1.5%) UDP-GalNAc (280)74 (1.7%) 19 (2.9%) UDP-GalA (150) 58 (1.4%) 19 (2.9%) UDP-GlcNAc + EDTA31 (0.7%) — UDP-GlcA + EDTA — 22 (3.4%)*Membranes (324 ng protein) were incubated at 37° C. for 1 h with either120 μM UDP-[¹⁴C]GlcA (2.8 × 10⁴ dpm) or 300 μM UDP-[³H]GlcNAc (2 × 10⁴dpm). The radiolabeled sugar nucleotide was used in the presence of theindicated second nonlabeled sugar nucleotide. HA synthase activity wasdetermined as described in the application.

The term “modified structure” as used herein denotes a hyaluronic acidpolymer containing a sugar or derivative not normally found in thenaturally occurring HA polysaccharide. The term “modified sizedistribution” refer to the synthesis of hyaluronic acid molecules of asize distribution not normally found with the native enzyme; theengineered size could be much smaller or larger than normal.

Various hyaluronic acid products of differing size have application inthe areas of drug delivery and the generation of an enzyme of alteredstructure can be combined with a hyaluronic acid of differing size.Applications in angiogenesis and wound healing are potentially large ifhyaluronic acid polymers of about 20 monosaccharides can be made in goodquantities. Another particular application for small hyaluronic acidoligosaccharides is in the stabilization of recombinant human proteinsused for medical purposes. A major problem with such proteins is theirclearance from the blood and a short biological half life. One presentsolution to this problem is to couple a small molecule shield thatprevents the protein from being cleared from the circulation toorapidly. Very small molecular weight hyaluronic acid is well suited forthis role and would be nonimmunogenic and biocompatible. Largermolecular weight hyaluronic acid attached to a drug or protein may beused to target the reticuloendothelial cell system which has endocyticreceptors for hyaluronic acid.

One of ordinary skill in the art given this disclosure would appreciatethat there are several ways in which the size distribution of thehyaluronic acid polymer made by the hyaluronate synthase could beregulated to give different sizes. First, the kinetic control of productsize can be altered by decreasing temperature, decreasing time of enzymeaction and by decreasing the concentration of one or both sugarnucleotide substrates. Decreasing any or all of these variables willgive lower amounts and smaller sizes of hyaluronic acid product. Thedisadvantages of these approaches are that the yield of product willalso be decreased and it may be difficult to achieve reproducibilityfrom day to day or batch to batch.

Secondly, the alteration of the intrinsic ability of the enzyme tosynthesize a large hyaluronic acid product. Changes to the protein canbe engineered by recombinant DNA technology, including substitution,deletion and addition of specific amino acids (or even the introductionof prosthetic groups through metabolic processing). Such changes thatresult in an intrinsically slower enzyme could then allow morereproducible control of hyaluronic acid size by kinetic means. The finalhyaluronic acid size distribution is determined by certaincharacteristics of the enzyme, that rely on particular amino acids inthe sequence. Among the 20% of residues absolutely conserved between thestreptococcal enzymes and the eukaryotic hyaluronate synthases, there isa set of amino acids at unique positions that control or greatlyinfluence the size of the hyaluronic acid polymer that the enzyme canmake. Specific changes in any of these residues can produce a modifiedHAS that produces an HA product having a modified size distribution.Engineered changes to seHAS, spHAS, pmHAS, or cvHAS that decrease theintrinsic size of the hyaluronic acid that the enzyme can make beforethe hyaluronic acid is released, will provide powerful means to producehyaluronic acid product of smaller or potentially larger size than thenative enzyme.

Finally, larger molecular weight hyaluronic acid made be degraded withspecific hyaluronidases to make lower molecular weight hyaluronic acid.This practice, however, is very difficult to achieve reproducibility andone must meticulously repurify the hyaluronic acid to remove thehyaluronidase and unwanted digestion products.

As shown in FIG. 4, hyaluronan synthase can be engineered to producehyaluronic acid polymers of different size, in particular smaller, thanthe normal wildtype enzyme. The figure shows the distribution of HAsizes (in millions of Daltons, a measure of molecular weight) for aseries of spHAS enzymes, each of which was engineered by site directedmutagenesis to have a single amino acid change from the native enzyme.Each has a different Cysteine residue replaced with Alanine. The clusterof five curves with open symbols represent the following spHAS proteins:wildtype, C124A, C261A, C366A, and C402A. The filled circles representthe poorly expressed C225A protein which is only partially active.

The filled triangles is the C280A spHAS protein, which is found tosynthesize a much smaller range of HA polymers than the normal enzyme orthe other variants shown. This reduction to practice shows that it isfeasible to engineer the hyaluronate synthase enzyme to synthesize adesired range of HA product sizes. The seHAS, pmHAS, and cvHAS genesencoding hyaluronate synthase can also be manipulated by site directedmutagenesis to produce an enzyme which synthesizes a desired range of HAproduct sizes.

Structurally modified hyaluronic acid is no different conceptually thanaltering the size distribution of the hyaluronic acid product bychanging particular amino acids in the desired HAS or the spHAS.Derivatives of UDP-GlcNAc, in which the N-acetyl group is missingUDP-GlcN or replaced with another chemically useful group, are expectedto be particularly useful. The strong substrate specificity must rely ona particular subset of amino acids among the 20% that are conserved.Specific changes to one or more of these residues creates a functionalsynthase that interacts less specifically with one or more of thesubstrates than the native enzyme. This altered enzyme could thenutilize alternate natural or special sugar nucleotides to incorporatesugar derivatives designed to allow different chemistries to be employedfor the following purposes: (i) covalently coupling specific drugs,proteins, or toxins to the structurally modified hyaluronic acid forgeneral or targeted drug delivery, radiological procedures, etc., (ii)covalently cross linking the hyaluronic acid itself or to other supportsto achieve a gel, or other three dimensional biomaterial with strongerphysical properties, and (iii) covalently linking hyaluronic acid to asurface to create a biocompatible film or monolayer.

Bacteria can also be engineered to produce hyaluronic acid. Forinstance, we have created strains of B. subtilis containing the spHASgene, as well as the gene for one of the sugar nucleotide precursors. Wechose this bacteria since it is frequently used in the biotech industryfor the production of products for human use. These bacteria wereintended as first generation prototypes for the generation of abacterium able to produce hyaluronic acid in larger amounts thanpresently available using a wild type natural strain. We put in multiplecopies of these genes.

For example, three Bacillus subtilis strains were constructed to containone or both of the Streptococcus pyogenes genes for hyaluronan synthase(spHAS) and UDP-glucose dehydrogenase, the results of which are shown inTable II-B. Based on a sensitive commercial radiometric assay to detectand quantitate HA, it was determined that the strain with both genes(strain #3) makes and secretes HA into the medium. The parent strain orthe strain with just the dehydrogenase gene (strain #1) does not makeHA. Strain #2, which contains just the spHAS gene alone makes HA, butonly 10% of what strain #3 makes. Agarose gel electrophoresis showedthat the HA secreted into the medium by strain #3 is very high molecularweight. TABLE II-B Strain with Cell density Strain Number CellsMedium(*) genes (A₆₀₀) (μg HA per ml of culture) 1 0 0 hasB 4.8 2 4 35SpHAS 3.9 3 =>10 >250 SpHAS + hasB 3.2(*)Most HA is in media but some was cell-associated; HA was determinedusing the HA Test 50 kit from Pharmacia.

These experiments used the streptococcal promoters normally found withthese genes to drive protein expression. It is expected that theconstruction of strains with the spHAS or seHAS reading frame undercontrol of a B. subtilis promoter would yield even more superiorresults. The vector used is a Gram positive/E. coli shuttle vector thathas a medium copy number in B. subtilis and a gene for erythromycinresistance (enabling resistence to 8 μg/ml in B. subtilis or 175 μg/mlin E. coli). The B. subtilis host strain used is 1A1 from BGSC, whichhas a tryptophan requirement but otherwise is wildtype, and cansporulate. Cell growth and HA production was in Spizizens Minimal Mediaplus tryptophan, glucose, trace elements and erthromycin (8 μg/ml).Growth was at 32 degrees Celsius with vigorous agitation until themedium was exhausted (˜36 hours).

This demonstrates that these bioengineered cells, which would notnormally make hyaluronic acid, became competent to do so when they aretransformed with the spHAS gene. The seHAS would also be capable ofbeing introduced into a non-hyaluronic acid producing bacteria to createa bioengineered bacterial strain capable of producing hyaluronic acid.

A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to an HAS protein composition, wherein the HAS proteinor appropriately modified HAS protein (e.g., containing a [HIS]₆ tail)is purified to any degree relative to its naturally-obtainable state,i.e., in this case, relative to its purity within a prokaryotic cellextract. HAS protein may be isolated from Streptococcus, Pasteurella,Chlorella virus, patient specimens, recombinant cells, infected tissues,isolated subpopulation of tissues that contain high levels ofhyaluronate in the extracellular matrix, and the like, as will be knownto those of skill in the art, in light of the present disclosure. Forinstance, the recombinant seHAS or spHAS protein makes up approximately10% of the total membrane protein of E. coli. A purified HAS proteincomposition therefore also refers to a polypeptide having the amino acidsequence of SEQ ID NO:2, free from the environment in which it maynaturally occur (FIG. 5).

Turning to the expression of the seHAS gene whether from genomic DNA, ora cDNA, one may proceed to prepare an expression system for therecombinant preparation of the HAS protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression.

HAS may be successfully expressed in eukaryotic expression systems,however, the inventors aver that bacterial expression systems can beused for the preparation of HAS for all purposes. It is believed thatbacterial expression will ultimately have advantages over eukaryoticexpression in terms of ease of use, cost of production, and quantity ofmaterial obtained thereby.

The purification of streptococcal hyaluronan synthase (seHAS and spHAS)is shown in Table III and FIG. 6. Fractions from various stages of thepurification scheme were analyzed by SDS-PAGE on a 12.5% gel, which wasthen stained with Coomassie Brilliant Blue R-250. Lanes: molecularweight markers; 1, whole E. coli membranes containing the recombinantseHAS-H6; 2, insoluble fraction after detergent solubilization ofmembranes; 3, detergent solubilized fraction; 4, flow-through from theNi-NTA chromatography resin; 5-9, five successive washes of the column(two column volumes each); 10, the eluted pure HA synthase which is asingle band. TABLE III Total Specific Total Activity Purifi- ProteinActivity (nmol UDP- Yield cation Step (μg) (mmol/μg/hr) GlcA) (%)(-fold) Membranes 3690 1.0 3649 100 1.0 Extract 2128 2.2 4725 129 2.2Affinity 39 13 500 14 13.1 Column

It is proposed that transformation of host cells with DNA segmentsencoding HAS will provide a convenient means for obtaining a HASprotein. It is also proposed that cDNA, genomic sequences, andcombinations thereof, are suitable for eukaryotic expression, as thehost cell will, of course, process the genomic transcripts to yieldfunctional mRNA for translation into protein.

Another embodiment of the present invention is a method of preparing aprotein composition comprising growing a recombinant host cellcomprising a vector that encodes a protein which includes an amino acidsequence in accordance with SEQ ID NO:2 or functionally similar withconserved or semi-conserved amino acid changes. The host cell will begrown under conditions permitting nucleic acid expression and proteinproduction followed by recovery of the protein so produced. Theproduction of HAS and ultimately HA, including the host cell, conditionspermitting nucleic acid expression, protein production and recovery willbe known to those of skill in the art in light of the present disclosureof the seHAS gene, and the seHAS gene protein product HAS, and by themethods described herein.

Preferred hosts for the expression of hyaluronic acid are prokaryotes,such as S. equisimilis, and other suitable members of the Streptococcusspecies. However, it is also known that HA may be synthesized byheterologous host cells expressing recombinant HA synthase, such asspecies members of the Bacillus, Enterococcus, or even Escherichiagenus. A most preferred host for expression of the HA synthase of thepresent invention is a bacteria transformed with the HAS gene of thepresent invention, such as Lactococcus species, Bacillus subtilis or E.coli.

It is similarly believed that almost any eukaryotic expression systemmay be utilized for the expression of HAS e.g., baculovirus-based,glutamine synthase-based, dihydrofolate reductase-based systems, SV40based, adenovirus-based, cytomegalovirus-based, yeast-based, and thelike, could be employed. For expression in this manner, one wouldposition the coding sequences adjacent to and under the control of thepromoter. It is understood in the art that to bring a coding sequenceunder the control of such a promoter, one positions the 5′ end of thetranscription initiation site of the transcriptional reading frame ofthe protein between about 1 and about 50 nucleotides “downstream” of(i.e., 3′ of) the chosen promoter. Also, Saccharomyces cevevisiae yeastexpression vector systems, such as pYES2, will also produce HAS undercontrol of the GAL promoter as shown in FIG. 7. FIG. 7 shows that thespHAS enzyme was produced in recombinant yeast using the pYES2 plasmid.When supplied with UDP-GlcA and UDP-GlcNAc, the enzyme makes highmolecular weight HA.

Where eukaryotic expression is contemplated, one will also typicallydesire to incorporate into the transcriptional unit which includes theHAS gene or DNA, an appropriate polyadenylation site (e.g.,5′-AATAAA-3′) if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides “downstream” of the termination site of the protein at aposition prior to transcription termination.

It is contemplated that virtually any of the commonly employed hostcells can be used in connection with the expression of HAS in accordanceherewith. Examples of preferred cell lines for expressing HAS cDNA ofthe present invention include cell lines typically employed foreukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI38, BHK, COS-7, RIN and MDCK cell lines. This will generally include thesteps of providing a recombinant host bearing the recombinant DNAsegment encoding the HAS enzyme and capable of expressing the enzyme;culturing the recombinant host in media under conditions that will allowfor transcription of the cloned HAS gene or cDNA and appropriate for theproduction of the hyaluronic acid; and separating and purifying the HASenzyme or the secreted hyaluronic acid from the recombinant host.

Generally, the conditions appropriate for expression of the cloned HASgene or cDNA will depend upon the promoter, the vector, and the hostsystem that is employed. For example, where one employs the lacpromoter, one will desire to induce transcription through the inclusionof a material that will stimulate lac transcription, such asisopropylthiogalactoside. For example, the cloned seHAS gene of thepresent invention is expressed as a HIS₆ containing protein in E. colias shown in FIG. 5. Where other promoters are employed, differentmaterials may be needed to induce or otherwise up-regulatetranscription.

FIG. 5 depicts the overexpression of recombinant seHAS and spHAS in E.coli. Membrane proteins (5 mg per lane) were fractionated by SDS-PAGEusing a 10% (w/v) gel under reducing conditions. The gel was stainedwith Coomassie blue R-250, photographed, scanned, and quantitated usinga molecular dynamics personal densitometer (model PDSI P60). Theposition of HA synthase is marked by the arrow. Lane A is native spHAS(Group A); Lane C is native seHAS; Lane E is recombinant seHAS; Lane Pis recombinant spHAS; Lane V is vector alone. Standards used wereBio-rad low M_(r) and shown in kDa.

In addition to obtaining expression of the synthase, one will preferablydesire to provide an environment that is conducive to HA synthesis byincluding appropriate genes encoding enzymes needed for the biosynthesisof sugar nucleotide precursors, or by using growth media containingsubstrates for the precursor-supplying enzymes, such asN-acetylglucosamine or glucosamine (GlcNAc or GlcNH₂) and glucose (Glc).

One may further desire to incorporate the gene in a host which isdefective in the enzyme hyaluronidase, so that the product synthesizedby the enzyme will not be degraded in the medium. Furthermore, a hostwould be chosen to optimize production of HA. For example, a suitablehost would be one that produced large quantities of the sugar nucleotideprecursors to support the HAS enzyme and allow it to produce largequantities of HA. Such a host may be found naturally or may be made by avariety of techniques including mutagenesis or recombinant DNAtechnology. The genes for the sugar nucleotide synthesizing enzymes,particularly the UDP-Glc dehydrogenase required to produce UDP-GlcA,could also be isolated and incorporated in a vector along with the HASgene or cDNA. A preferred embodiment of the present invention is a hostcontaining these ancillary recombinant gene or cDNAs and theamplification of these gene products thereby allowing for increasedproduction of HA.

The means employed for culturing of the host cell is not believed to beparticularly crucial. For useful details, one may wish to refer to thedisclosure of U.S. Pat. Nos. 4,517,295; 4,801,539; 4,784,990; or4,780,414; all incorporated herein by reference. Where a prokaryotichost is employed, such as S. equisimilis, one may desire to employ afermentation of the bacteria under anaerobic conditions in CO₂-enrichedbroth growth media. This allows for a greater production of HA thanunder aerobic conditions. Another consideration is that Streptococcalcells grown anaerobically do not produce pyrogenic exotoxins.Appropriate growth conditions can be customized for other prokaryotichosts, as will be known to those of skill in the art, in light of thepresent disclosure.

Once the appropriate host has been constructed, and cultured underconditions appropriate for the production of HA, one will desire toseparate the HA so produced. Typically, the HA will be secreted orotherwise shed by the recombinant organism into the surrounding media,allowing the ready isolation of HA from the media by known techniques.For example, HA can be separated from the cells and debris by filteringand in combination with separation from the media by precipitation byalcohols such as ethanol. Other precipitation agents include organicsolvents such as acetone or quaternary organic ammonium salts such ascetyl pyridinium chloride (CPC).

A preferred technique for isolation of HA is described in U.S. Pat. No.4,517,295, and which is incorporated herein by reference, in which theorganic carboxylic acid, trichloroacetic acid, is added to the bacterialsuspension at the end of the fermentation. The trichloroacetic acidcauses the bacterial cells to clump and die and facilitates the ease ofseparating these cells and associated debris from HA, the desiredproduct. The clarified supernatant is concentrated and dialyzed toremove low molecular weight contaminants including the organic acid. Theaforementioned procedure utilizes filtration through filter cassettescontaining 0.22 μm pore size filters. Diafiltration is continued untilthe conductivity of the solution decreases to approximately 0.5mega-ohms.

The concentrated HA is precipitated by adding an excess of reagent gradeethanol or other organic solvent and the precipitated HA is then driedby washing with ethanol and vacuum dried, lyophilized to remove alcohol.The HA can then be redissolved in a borate buffer, pH 8, andprecipitated with CPC or certain other organic ammonium salts such asCETAB, a mixed trimethyl ammonium bromide solution at 4 degree(s)Celsius. The precipitated HA is recovered by coarse filtration,resuspended in 1 M NaCl, diafiltered and concentrated as furtherdescribed in the above referenced patent. The resultant HA is filtersterilized and ready to be converted to an appropriate salt, dry powderor sterile solution, depending on the desired end use.

A. Typical Genetic Engineering Methods which May be Employed

If cells without formidable cell membrane barriers are used as hostcells, transfection is carried out by the calcium phosphateprecipitation method, well known to those of skill in the art. However,other methods may also be used for introducing DNA into cells such as bynuclear injection, cationic lipids, electroporation, protoplast fusionor by the Biolistic™ Bioparticle delivery system developed by DuPont(1989). The advantage of using the DuPont system is a hightransformation efficiency. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, the preferred method oftransfection is calcium treatment using calcium chloride to inducecompetence or electroporation.

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to construct the plasmids required. Cleavage is performed bytreating with restriction enzyme (or enzymes) in suitable buffer. Ingeneral, about 1 μg plasmid or DNA fragments are used with about 1 unitof enzyme in about 20 μl of buffer solution. Appropriate buffers andsubstrate amounts for particular restriction enzymes are specified bythe manufacturer. Incubation times of about 1 hour at 37° C. areworkable.

After incubations, protein is removed by extraction with phenol andchloroform, and the nucleic acid is recovered from the aqueous fractionby precipitation with ethanol. If blunt ends are required, thepreparation is treated for 15 minutes at 15° C. with 10 units ofPolymerase I (Klenow), phenol-chloroform extracted, and ethanolprecipitated. For ligation approximately equimolar amounts of thedesired components, suitably end tailored to provide correct matchingare treated with about 10 units T4 DNA ligase per 0.5 μg DNA. Whencleaved vectors are used as components, it may be useful to preventreligation of the cleaved vector by pretreatment with bacterial alkalinephosphatase.

For analysis to confirm functional sequences in plasmids constructed,the first step was to amplify the plasmid DNA by cloning intospecifically competent E. coli SURE cells (Stratagene) by doingtransformation at 30-32° C. Second, the recombinant plasmid is used totransform E. coliK5 strain Bi8337-41, which can produce the UDP-GlcAprecursor, and successful transformants selected by antibioticresistance as appropriate. Plasmids from the library of transformantsare then screened for bacterial colonies that exhibit HA production.These colonies are picked, amplified and the plasmids purified andanalyzed by restriction mapping. The plasmids showing indications of afunctional HAS gene are then further characterized by any number ofsequence analysis techniques which are known by those of ordinary skillin the art.

B. Source and Host Cell Cultures and Vectors

In general, prokaryotes were used for the initial cloning of DNAsequences and construction of the vectors useful in the invention. It isbelieved that a suitable source may be Gram-positive cells, particularlythose derived from the Group C Streptococcal strains. Bacteria with asingle membrane, but a thick cell wall such as Staphylococci andStreptococci are Gram-positive. Gram-negative bacteria such as E. colicontain two discrete membranes rather than one surrounding the cell.Gram-negative organisms tend to have thinner cell walls. The singlemembrane of the Gram-positive organisms is analogous to the inner plasmamembrane of Gram-negative bacteria. The preferred host cells areStreptococcus strains that are mutated to become hyaluronidase negativeor otherwise inhibited (EP144019, EP266578, EP244757). Streptococcusstrains that have been particularly useful include S. equisimilis and S.zooepidemicus.

Prokaryotes may also be used for expression. For the expression of HAsynthase in a form most likely to accommodate high molecular weight HAsynthesis, one may desire to employ Streptococcus species such as S.equisimilis or S. zooepidemicus. The aforementioned strains, as well asE. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli suchas Bacillus subtilis, or other enterobacteriaceae such as Serratiamarcescens, could be utilized to generate a “super” HAS containing host.

In general, plasmid vectors containing origins of replication andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries an origin of replication, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species. pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. A pBR plasmid or a pUC plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

Those promoters most commonly used in recombinant DNA constructioninclude the lacZ promoter, tac promoter, the T7 bacteriophage promoter,and tryptophan (trp) promoter system. While these are the most commonly:used, other microbial promoters have been discovered and utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally with plasmidvectors. Also for use with the present invention one may utilizeintegration vectors.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used. Thisplasmid already contains the trp1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow withouttryptophan, for example, ATCC No. 44076 or PEP4-1. The presence of thetrp1 lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan. Suitable promoting sequences in yeastvectors include the promoters for the galactose utilization genes, the3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes are also ligated into the expression vector3′ of the sequence desired to be expressed to provide polyadenylation ofthe mRNA and termination. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions are thepromoter region for alcohol dehydrogenase 2, cytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Any plasmidvector containing a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture has become a routineprocedure in recent years. Examples of such useful host cell lines areVERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138,BHK, COS, and MDCK cell lines.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, bovine papilloma virusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the Hind III site toward the Bg1/site located in the viral originof replication.

Further, it is also possible, and often desirable, to utilize promoteror control sequences normally associated with the desired gene sequence,provided such control sequences are compatible with the host cellsystems. An origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter mechanismis often sufficient.

C. Isolation of a Bona Fide HA Synthase Gene from a Highly EncapsulatedStrain of Group C Streptococcus equisimilis.

The encoded protein, designated seHAS, is 417 amino acids (calculatedmolecular weight of 47,778 and pl of 9.1) and is the smallest member ofthe HAS family identified thus far (FIG. 2). seHAS also migratesanomalously fast in SDS-PAGE (M_(r)˜42 kDa) (FIGS. 5 and 8).

FIG. 8 is a graphical representation of a Western Blot analysis ofrecombinant seHAS using specific antibodies. Group C(C; lane 1) or GroupA (A; lane 4) Streptococcal membranes and E. coli membranes (9 mg/lane)containing recombinant seHAS (E; lanes 2, 7, and 9) or spHAS (P; lanes3, 6, 8, and 10) were fractionated by reducing SDS-PAGE andelectrotransferred to nitrocellulose. Strips of nitrocellulose wereprobed and developed as described in the application using purified IgGfractions raised to the following regions of spHAS: central domainpeptide E¹⁴⁷-T¹⁶¹ (lanes 14); C-terminus peptide (lanes 5-6); thecomplete protein (lanes 7 and 8); recombinant central domain (lanes 9and 10). Nonimmune IgG or membranes from cells transformed with vectoralone gave no staining as in lane 5.

The seHAS and spHAS protein (previously identified in U.S. Ser. No.08/899,940) encoding sequences are 72% identical. The deduced proteinsequence of seHAS was confirmed by reactivity with a synthetic peptideantibody (FIG. 8). Recombinant seHAS expressed in E. coli was recoveredin membranes as a major protein (FIG. 5) and synthesized very largemolecular weight HA in the presence of UDP-GlcNAc and UDP-GlcA in vitro(FIG. 9).

FIG. 9 shows a kinetic analysis of the HA size distributions produced byseHAS and spHAS. E. coli membranes containing equal amounts of seHAS orspHAS protein were incubated at 37° C. with 1.35 mM UDP-[¹⁴C] GlCA(1.3×10³ dpm/nmol) and 3.0 mM UDP-GlcNAc as described in theapplication. These substrate concentrations are greater than 15 timesthe respective Km valves. Samples taken at 0.5, 1.0, and 60 min weretreated with SDS and chromatographed over Sephacryl S400 HR. The HAprofiles in the fractionation range of the column (fractions 12-24) arenormalized to the percent of total HA in each fraction. The values abovethe arrows in the top panel are the MWs (in millions) of HA determineddirectly in a separate experiment using a Dawn multiangle laser lightscattering instrument (Wyatt Technology Corp.). The size distributionsof HA synthesized by seHAS (●, ▪, ▴) and spHAS (◯, □, _) at 0.5 min (◯,●), 1.0 min (□, ▪) and 60 min (_, ▴) are shown as indicated. Analysisshowed that seHAS and spHAS are essentially identical in the sizedistribution of HA chains they synthesize (FIG. 9). SeHAS is twice asfast as spHAS in its ability to make HA.

C.1 Bacterial Strains and Vectors

The mucoid group C strain D181; (Streptococcus equisimilis) was obtainedfrom the Rockefeller University Collection. The E. coli host strainsSure and XL1-Blue MRF′ were from Stratagene and strain Top10 F′ was fromInvitrogen. Unless otherwise noted, Streptococci were grown in THY andE. coli strains were grown in LB medium. pKK-223 Expression vector wasfrom Pharmacia, PCR 2.1 cloning vector was from Invitrogen, andpredigested A Zap Express™ Bam HI/CIAP Vector was from Stratagene.

C.2 Recombinant DNA and Cloning

High molecular mass Genomic DNA from Streptococcus equisimilis isolatedby the method of Caparon and Scott (as known by those with ordinaryskill in the art) was partially digested with Sau3A1 to an average sizeof 2-12 kb. The digested DNA was precipitated with ethanol, washed andligated to the Bam HI/CIAPλ Zap Express vector. Ligated DNA was packagedinto phage with a Packagene™ extract obtained from Promega. The titer ofthe packaged phage library was checked using XL1-Blue MRF′ E. coli as ahost.

C.3 Degenerate PCR Amplification

Degenerate oligonucleotides were designed based upon conserved sequencesamong spHAS (Streptococcus pyogenes), DG42 (Xenopus laevis HAS; 19) andnodC (a Rhizobium meliloti nodulation factor; 20) and were used for PCRamplification with D181 genomic DNA as a template. Amplificationconditions were 34 cycles at: 94° C. for 1 min, 44° C. for 1 min, 72° C.for 1.5 min followed by a final extension at 72° C. for 10 min.Oligonucleotide HADRF1, 5′-GAY MGA YRT YTX ACX AAT TAY GCT ATH GAY TTRGG-3′ (sense strand) corresponds to the sequence D²⁵⁹RCLTNYAIDL (spHAS).Oligonucleotide HACTR1, 5′-ACG WGT WCC CCA NTC XGY ATT TTT NAD XGTRCA-3′ (antisense strand) corresponds to the region C⁴⁰⁴TIKNTEWGTR(spHAS). The degeneracy of bases at some positions are represented bynomenclature adopted by the IUPAC in its codes for degenerate baseslisted in Table IV. TABLE IV IUPAC Codes - Degenerate Bases TheInternational Union for Pure and Applied Chemistry (IUPAC) hasestablished a standard single-letter designation for degenerate bases.These are: B = C + G + T D = A + G + T H = A + C + T K = T + G M = A + CN = A + C + G + T R = A + G S = G + C W = A + T V = A + C + G X = aminor bases (specified elsewhere) Y = C + T

These two oligonucleotides gave a 459 bp PCR product, which wasseparated on an agarose gel and purified using the BIO-101 Genecleankit. This fragment was then cloned into PCR2.1 vector using TOP 10 F′cells as a host according to the manufacturer's directions. Doublestranded plasmid DNA was purified from E. coli (Top 10 F′) using theQIAfilter Plasmid Midi Kit (Qiagen). Two other degenerate sense primerswere also synthesized: HAVAF1, 5′-GTN GCT GCT GTW RTX CCW WSX TWT AAYGAR GA-3′ (corresponding to the region V⁶⁶AAVIPSYNE of spHAS) andHAVDF1, 5′-GTX RWT GAY GGN WSX WSN RAX GAT GAX GC-3′ (based onV¹⁰⁰DDGSSNTD of spHAS). Two unique antisense primers were synthesizedbased on the sequence of the 459 bp PCR product. These were: D181.2,5′-GAA GGA CTT GTT CCA GCG GT-3′ and D181.4, 5′-TGA ATG TTC CGA CAC AGGGC-3′. Each of the two degenerate sense primers, when used with eitherD181.2 or D181.4 to amplify D181 genomic DNA, gave expected size PCRproducts. The four PCR products were cloned and sequenced using the samestrategy as above. For each PCR product, sequences obtained from sixdifferent clones were compared in order to derive a consensus sequence.Thus we obtained a 1042 bp sequence with a continuous ORF with highhomology to spHAS.

C.4 Library Screening

Two molecular probes were used to screen the library; the cloned 459 bpPCR product and oligonucleotide D181.5 (5′-GCTTGATAGGTCACCAGTGTCACG-3′;derived from the 1042 bp sequence). The 459 bp PCR product wasradiolabeled using the Prime-It 11 random primer labeling Kit(Stratagene) according to the manufacturers instructions.Oligonucleotides were labeled by Kinace-It Kinasing Kit (Stratagene)using [γ³²P]ATP. Radiolabeled products were separated from nonlabeledmaterial on NucTrap Push columns (Stratagene). The oligoprobe hybridizedspecifically with a D181 genomic digest on Southern blots. To screen theA phage library, XLBLUE MRF′ was used as a host (3000 plaques/plate) onNitrocellulose membranes containing adsorbed phage, were prehybridizedat 60° C. and hybridized with 5′-end labeled oligonucleotide, D181.5, inQuikHyb Hybridization solution (Stratagene) at 80° C. according toinstructions.

The membranes were then washed with 2×SSC buffer and 0.1% (w/v) SDS atroom temperature for 15 min, at 60° C. with 0.1×SSC buffer and 0.1% SDS(w/v) for 30 min, dried and then exposed to Bio-Max MS film overnight at−70° C. Positive plaques were replated and rescreened twice. Purepositive phages were saved in SM buffer with chloroform. PCR on thesephages with vector primers revealed 3 different insert sizes.

PCR with a combination of vector primers and primers from differentregions of the cloned 1042 bp sequence revealed that only one of thethree different phages had the complete HAS gene. The insert size inthis phage was 6.5 kb. Attempts to subclone the insert into plasmid formby autoexcision from the selected phage library clone failed. Therefore,a PCR strategy was applied again on the pure positive phage DNA toobtain the 5′ and 3′ end of the ORF. Oligonucleotide primers D181.3(5′-GCCCTGTGTCGGAACATTCA-3′) and T3 (vector primer) amplified a 3 kbproduct and oligonucleotides D181.5 and T7 (vector primer) amplified a2.5 kb product. The 5′ and 3′-end sequences of the ORF were obtained bysequencing these two above products. Analysis of all PCR productsequences allowed us to reconstruct the ORF of the 1254 bp seHAS gene.

C.5 Expression Cloning of the seHAS

Primers were designed at the start and stop codon regions of seHAS tocontain an EcoR1 restriction site in the sense oligonucleotide(5′-AGGATCCGAATTCATGAGAACATTAAAAAACCTC-3′) and a Pst1 site in theantisense oligonucleotide (5′-AGAATTCTGCAGTTATAATAATTTTTTACGTGT-3′).These primers amplified a 1.2 kb PCR product from D181 genomic DNA aswell as from pure hybridization-positive phage. The 1.2 kb product waspurified by agarose gel electrophoresis, digested with Pst1 and EcoR1and cloned directionally into Pst1- and EcoR1-digested pKK223 vector.The ligated vector was transformed into E. coli SURE cells that werethen grown at 30° C. This step was practically important since otherhost cells or higher temperatures resulted in deletions of the clonedinsert. Colonies were isolated and their pDNA purified. Out of sixcolonies (named a, b, c, d, e, and f), five had the correct size insert,while one had no insert.

C.6 HA Synthase Activity

HA synthase activity was assayed in membranes prepared from the 5 aboveclones. Fresh log phase cells were harvested at 3000 g, washed at 4° C.with PBS and membranes were isolated by a modification of a protoplastmethod as known by those of ordinary skill in the art. Membranepreparations from Streptococcus pyogenes and Streptococcus equisimiliswere also obtained by modification of a different protoplast procedure.Membranes were incubated at 37° C. in 50 mM sodium and potassiumphosphate, pH 7.0 with 20 mM MgCl₂, 1 mM DTE, 120 μM UDP-GlcA and 300 μMUDP-GlcNAc. Incorporation of sugar was monitored by using UDP-[¹⁴C]GicA(318 mCi/mmol; ICN) and/or UDP-[³H]GlcNAc (29.2 Ci/mmol NEN). Reactionswere terminated by addition of SDS to a final concentration of 2% (w/v).Product HA was separated from precursors by descending paperchromatography and measured by determining incorporated radioactivity atthe origin.

C.7 Gel Filtration Analysis

Radiolabeled HA produced in vitro by membranes containing recombinantseHAS or spHAS was analyzed by chromatography on a column (0.9×40 cm) ofSephacryl S500 HR (Pharmacia Biotech Inc.). Samples (0.4 ml in 200 mMNaCl, 5 mM Tris-HCl, pH 8.0, plus 0.5% SDS) were eluted with 200 mM,NaCl, 5 mM Tris-HCl, and pH 8.0 and 0.5 ml fractions were assessed for¹⁴C and/or ³H radioactivity. Authenticity of the HA polysaccharide wasassessed by treatment of a separate identical sample with theHA-specific hyaluronate lyase of Streptomyces hyalurolyticus (EC4.2.2.1) at 37° C. for 3 hrs. The digest was then subjected to gelfiltration.

C.8 SDS-PAGE and Western Blotting

SDS-PAGE was performed according to the Laemmli method. Electrotransfersto nitrocellulose were performed within standard blotting buffer with20% methanol using a Bio-Rad mini Transblot device. The blots wereblocked with 2% BSA in TBS. Protein A/G alkaline phosphatase conjugate(Pierce) and p-nitroblue tetrazolium/5-bromo-4-chloro-3 indolylphosphate p-toluidine salt were used for detection.

C.9 DNA Sequence and Analysis

Plasmids were sequenced on both strands using fluorescent labeled vectorprimers. Sequencing reactions were performed using a Thermosequenase™kit for fluorescent labeled primers (with 7-deazaG). Samples wereelectrophoresed on a Pharmacia ALF Express DNA Sequencer and data wereanalyzed by the ALF Manager Software v3.02. Internal regions of insertswere sequenced with internal primers using the ABI Prism 377 (Softwareversion 2.1.1). Ambiguous regions were sequenced manually usingSequenase™ 7-deaza-DNA polymerase, 7-deaza GTP master mix (USB) and[α-³⁵S] dATP (Amersham Life Sciences). The sequences obtained werecompiled and analyzed using DNASIS, v2.1 (Hitachi Software EngineeringCo., Ltd.). The nucleotide and amino acid sequences were compared withother sequences in the Genbank and other databases.

C.10 Identification of seHAS

Identification of seHAS was accomplished by utilizing a PCR approachwith oligonucleotide primers based on several regions of high identityamong spHAS, DG42 (now known to be a developmentally regulated X. laevisHAS and designated xlHAS) and NodC (a Rhizobium β-GlcNAc transferase).The xlHAS and NodC proteins are, respectively, ˜50% and ˜10% identicalto spHAS. This strategy yielded a 459 bp PCR product whose sequence was66.4% identical to spHAS, indicating that a Group C homologue (seHAS) ofthe Group A (spHAS) HA synthase gene had been identified. The completecoding region of the gene was then reconstructed using a similarPCR-based strategy. A final set of PCR primers was then used to amplifythe complete ORF from genomic DNA. When this 1.2 kb PCR fragment wasincorporated into the expression vector pKK223 and transformed into E.coli SURE cells, HA synthetic activity was demonstrated in isolatedmembranes from 5 of the 5 colonies tested.

The ORF of the reconstructed gene encodes a novel predicted protein of417 amino acids that was not in the database and it is two amino acidsshorter than spHAS. The two bacterial proteins are 72% identical and thenucleic acid sequences are 70% identical. The predicted molecular weightof the seHAS protein is 47,778 and the predicted isoelectric point is atpH 9.1. Three recently identified mammalian HASs (muHAS1, muHAS2,muHAS3, FIG. 2) are similar to the bacterial proteins. The overallidentity between the two groups is ˜28-31%, and in addition many aminoacids in seHAS are highly conserved with those of the eukaryotic HASs(e.g., K/R or D/E substitutions). A98R, the PBCY-1 HAS is 28-33 percentidentical to the mammalian HASs, and is predicted to have a similartopology in the lipid membrane. Within mammalian species the same familymembers are almost completely identical (e.g., muHAS1 and huHAS1 are 95%identical; muHAS2 and huHAS2 are 98% identical). However, and as shownin FIG. 3, even within the same species the different HAS family membersare more divergent (e.g., muHAS1 and muHAS2 are 53% identical; muHAS1and muHAS3 are 57% identical; muHAS2 and muHAS3 are 71% identical).

FIG. 10 shows hydropathy plots for seHAS and predicted membranetopology. The hydrophilicity plot for the Streptococcal Group C HAS wasgenerated by the method of Kyte and Doolittle (J. Mol. Biol. 157, 105,1982) using DNASIS. The protein is predicted to be an integral membraneprotein.

FIG. 11 shows a model for the topologic organization of seHAS in themembrane. The proposed topology for the protein conforms to thecharge-in rule and puts the large central domain inside. This domain islikely to contain most of the substrate binding and catalytic functionsof the enzyme. Cys²²⁶ in seHAS, which is conserved in all HAS familymembers, as well as the other three cysteines are shown in the centraldomain. Cys²⁸¹ is a critical residue whose alteration can dramaticallyalter the size distribution of HA product synthesized by the enzyme.

The overall membrane topology predicted for seHAS is identical to thatfor spHAS and the eukaryotic HASs reported thus far. The protein has twoputative transmembrane domains at the amino terminus and 2-3membrane-associated or transmembrane domains at the carboxyl end. Thehydropathy plots for the two Streptococcal enzymes are virtuallyidentical and illustrate the difficulty in predicting the topology ofthe extremely hydrophobic region of ˜90 residues at K³¹³-R⁴⁰⁶ in seHAS(K³¹³-K⁴⁰⁵ in spHAS).

seHAS was efficiently expressed in E. coli cells. Roughly 10% of thetotal membrane protein was seHAS as assessed by staining of SDS-PAGEgels (FIG. 5). The prominent seHAS band at 42 kD is quantitativelymissing in the vector-only control lane. This unusually high level ofexpression for a membrane protein is also found for spHAS, using thesame vector in SURE cells. About 8% of the membrane protein is spHAS inE. coli SURE cells. In contrast, the amount of seHAS in Group Cmembranes is not more than 1% of the total membrane protein. The spHASin Group A membranes is barely detectable. The recombinant seHASexpressed in E. coli SURE cells does not synthesize HA in vivo, sincethese cells lack UDP-GlcA, one of the required substrates. Membranes,however containing the recombinant seHAS protein synthesize HA whenprovided with the substrates UDP-GlcNAc and UDP-GlcA (FIG. 12).

FIG. 12 shows the synthesis of authentic HA by recombinant seHAS. E.coli membranes (69 μg) prepare from cells containing recombinant seHASor vector alone were incubated at 37° C. for 1 hour with 700 μMUDP-[³H]GlcNAc (2.78×10³ dpm/nmol; □, ▪) and 300 μM UDP-[¹⁴C]GlcA(3.83×10³ dpm/nmol; ◯, ●) in a final volume of 200 μl as describedherein. The enzyme reaction was stopped by addition of EDTA to a finalconcentration of 25 mM. Half the reaction mix was treated withStreptomyces hyaluronidase at 37° C. for 3 hours. SDS (2%, w/v) wasadded to hyaluronidase-treated (◯, □) and untreated (●, ▪) samples,which were heated at 90° C. for 1 min. The samples were diluted to 500μl with column buffer (5 mM Tris, 0.2 M Nacl, pH 8.0), clarified bycentrifugation and 200 μl was injected onto a Sephacryl S-500 HR column.Fractions (1 ml) were collected and radioactivity was determined. BD isthe peak elution position of blue dextran (˜2×10⁶ DA; Pharmacia). V_(o)marks the excluded volume and V_(i) the included volume. The ratio of[¹⁴c] GlcA: [³H] GlcNAc incorporated into the total amount of HAfractionated on the column is 1.4, which is identical to the ratio ofspecific activities of the two substrates. Therefore, the molar ratiosof the sugars incorporated into product is 1:1 as predicted forauthentic HA. Membranes from cells transformed with vector alone did notsynthesize HA.

Using 120 μM UDP-GlcA and 300 μM UDP-GlcNAc, HA synthesis was linearwith membrane protein (at ≦0.2 μg) and for at least 1 hour. Also,membranes prepared from nontransformed cells or cells transformed withvector alone have no detectable HAS activity. HA synthesis is negligibleif Mg⁺² is chelated with EDTA (<5% of control) or if either of the twosubstrates are omitted (˜2% of control). Recombinant seHAS also showedthe expected specificity for sugar nucleotide substrates, being unableto copolymerize either UDP-GalA, UDP-Glc or UDP-GalNAc with either ofthe two normal substrates (Table II).

Based on gel filtration analysis, the average mass of the HA synthesizedby seHAS in isolated membranes is 5-10×10⁶ Da. The product of therecombinant seHAS is judged to be authentic HA based on the equimolarincorporation of both sugars and its sensitivity to degradation by thespecific Streptomyces hyaluronidase (FIG. 12). Although the conditionsfor total HA synthesis were not optimal (since ˜90% of one substrate wasincorporated into product), the enzyme produced a broad distribution ofHA chain lengths. The peak fraction corresponds to an HA mass of 7.5×10⁶Da which is a polymer containing approximately 36,000 monomeric sugars.The distribution of HA sizes resolved on this column ranged from2-20×10⁶ Da.

The deduced protein sequence of seHAS was confirmed by the ability ofantibodies to the spHAS protein to cross-react with the Group C protein(FIG. 8). Polyclonal antibodies to the whole spHAS protein or to justthe central domain of spHAS also reacted with the seHAS protein.Antipeptide antibody to the C-terminus of spHAS did not cross-react withthis somewhat divergent region in the seHAS protein. However,antipeptide antibody directed against the spHAS sequence E¹⁴⁷-T¹⁶¹recognized the same predicted sequence in seHAS. The antipeptideantibody also reacts with the native seHAS and spHAS proteins inStreptococcal membranes and confirms that the native and recombinantenzymes from both species are of identical size. Like the spHAS protein,seHAS migrates anomalously fast on SDS-PAGE. Although the calculatedmass is 47,778 Da, the M_(r) by SDS-PAGE is consistently ˜42 kDa.

Because of the sequence identity within their central domain regions andthe overall identical structure predicted for the two bacterial enzymes,the peptide-specific antibody against the region E¹⁴⁷-T¹⁶¹ can be usedto normalize for HAS protein expression in membranes prepared from cellstransformed with genes for the two different enzymes. Using thisapproach, membranes with essentially identical amounts of recombinantspHAS or seHAS were compared with respect to the initial rate of HAsynthesis and the distribution of HA product size.

As shown for spHAS, the synthesis of HA chains by seHAS is processive.The enzymes appear to stay associated with a growing HA chain until itis released as a final product. Therefore, it is possible to compare therates of HA elongation by seHAS and spHAS by monitoring the sizedistribution of HA chains produced at early times, during the firstround of HA chain synthesis. Based on gel filtration analysis of HAproduct sizes at various times, we estimated that the average rateelongation by seHAS is about 9,000 monosaccharides/minute at 37° C.(FIG. 9). In five minutes, the enzymes can polymerize an HA chain of5-10×10⁶ Da. During a 60 min incubation, therefore, each enzyme moleculecould potentially initiate, complete and release on the order of 5-8such large HA molecules. At early times (e.g., ≦1 min), reflectingelongation of the first HA chains, the size distribution of HA producedby seHAS was shifted to larger species compared to spHAS. By 60 min thetwo distributions of HA product sizes are indistinguishable.

The cloned seHAS represents the authentic Group C HA synthase.Previously reported or disclosed “Group C” proteins are, therefore, notthe true Group C HAS. The seHAS protein is homologous to nine of thecurrently known HA synthases from bacteria, vertebrates, and a virusthat now comprise this rapidly growing HA synthase family. This homologyis shown particularly in FIG. 2. In mammals three genes, designated HAS1, HAS 2 and HAS 3, have been identified and mapped to three differentchromosomes in both human and mouse. In amphibians the only HAS proteinidentified thus far is the developmentally regulated DG42, which wascloned in 1988 and recently shown to encode the HA synthase activity byanalysis of the recombinant protein in yeast membranes. Probably otherX. laevus HAS genes will soon be identified.

A divergent evolution model suggests that a primitive bacterial HASprecursor may have been usurped early during vertebrate development orthe bacterial pathogenic strategy of making an HA capsule was developedwhen a primitive bacteria captured in primordial HAS. Convergentevolution of the bacterial and eukaryotic HAS enzymes to a commonstructural solution seems unlikely, but may have occurred.

None of the three mammalian isozymes for HAS have yet been characterizedenzymatically with respect to their HA product size. At least tenidentified HAS proteins are predicted to be membrane proteins with asimilar topology. HA synthesis occurs at the plasma membrane and the HAis either shed into the medium or remains cell associated to form thebacterial capsule or a eukaryotic pericellular coat. The sugarnucleotide substrates in the cytoplasm are utilized to assemble HAchains that are extruded through the membrane to the external space.

The protein topology in the very hydrophobic carboxyl portion of the HASprotein appears to be critical in understanding how the enzymes extendthe growing HA chain as it is simultaneously extruded through themembrane. For example, the unprecedented enzymatic activity may requireunusual and complex interactions of the protein with the lipid bilayer.Preliminary results based on analysis of spHAS-alkaline phosphatesfusion proteins indicate that the amino and carboxyl termini and thelarge central domains are all intracellular, as shown in FIGS. 10 and11. The seHAS protein also contains a large central domain (˜63% of thetotal protein) that appears to contain the two substrate binding sitesand the two glycosyltransferase activities needed for HA synthesis.Although current software programs cannot reliably predict the number ornature of membrane-associated domains within the long C-terminalhydrophobic stretch, the proposed topological arrangement agrees withthe present evidence and applies as well to the eukaryotic enzymes,which are ˜40% larger primarily due to extension of the C-terminal endof the protein with 2 additional predicted transmembrane domains.

Four of the six Cys residues in spHAS are conserved with seHAs. OnlyCys225 in both bacterial enzymes is conserved in all members of the HASfamily. Since sulfhydryl reactive agents, such as p-mercurobenzoate orNEM, greatly inhibit HAS activity, it is likely that this conserved Cysis necessary or important for enzyme activity. Initial results fromsite-directed mutagenesis studies, however, indicate that a C225S mutantof spHAS is not inactive, it retains 5-10% of wildtype activity.

The recognition of nucleic acid sequences encoding only seHAS, onlyspHAS, or both seHAS and spHAS using specific oligonucleotides is shownin FIG. 13. Three pairs of sense-antisense oligonucleotides weredesigned based on the sequence of ID SEQ NO: 1 and the coding sequencefor spHAS. The seHAS based nucleic acid segments (se1-se2 andsesp1-sesp2) are listed in SEQ. ID NOS: 3, 4, 5, and 6. These threeoligonucleotide pairs were hybridized under typical PCR reactions withgenomic DNA from either Group C (seHAS) (lanes 2, 4, and 6) or Group A(spHAS) (lanes 3, 5, and 7) streptococci. Lanes 1 and 8 indicate thepositions of MW standards in kb (kilobases). The PCR reactions wereperformed using Taq DNA polymerase (from Promega) for 25 cycles asfollows: 94 degrees Celsius for 1 minute to achieve DNA denaturation, 48degrees Celsius (42 degrees Celsius for the smaller common sesp primers)for 1 minute to allow hybridization, and 72 degrees Celsius for 1.5minutes for DNA synthesis. The PCR reaction mixtures were then separatedby electrophoresis on a 1% agarose gel.

The se1-se2 primer pair was designed to be uniquely specific for theGroup C HAS (seHAS). The sp1-sp2 primer pair was designed to be uniquelyspecific for the Group A HAS (spHAS). The sesp1-sesp2 primer pair wasdesigned to hybridize to both the Group A and Group C HAS nucleic acidsequences. All three primer pairs behaved as expected, showing theappropriate ability to cross-hybridize and support the generation of PCRproducts that were specific and/or unique.

The oligonucleotides used for specific PCR or hybridization are listedin SEQ. ID NOS: 3, 4, 5, and 6. The synthetic oligonucleotides of SEQ IDNOS: 3, 4, 5, and 6 are indicated in the corresponding regions of SEQ IDNO:1. These regions are in bold face and marked, respectively as primersse1, se2, sesp1, and sesp2. The #1 indicates primers in the sensedirection, while the #2 indicates a primer in the antisense direction.Each of the four oligonucleotides will hybridize specifically with theseHAS sequence and the appropriate pairs of sense/antisense primers aresuitable for use in the polymerase chain reaction as shown in FIG. 13.

FIG. 7 shows a gel filtration analysis of hyaluronic acid synthesized byrecombinant HAS expressed in yeast membranes. A DNA fragment encodingthe open reading frame of 419 amino acid residues corresponding to spHAS(with the original Val codon switched to Met) was subcloned by standardmethods in the pYES2 yeast expression vector (from Invitrogen) toproduce pYES/HA. Membranes from cells with this construct were preparedby agitation with glass beads. The samples derived from pYES/HAconstructs contained substantial HA synthase activity and the “42 kDa”HAS protein was detected by Western analysis using specific antibodies;membranes from cells with vector alone possessed neither activity northe immunoreactive band (not shown). Membranes (315 ug protein) werefirst incubated with carrier free UDP-[¹⁴C]GlcA (1 uCi¹⁴C) amd 900 uMunlabeled UDP-GlcNAc in 50 mM Tris, pH 7, 20 mM MgCl₂, 1 mM DTT, and0.05 M NaCl (450 ul reaction volume) at 30 degrees Celsius for 1.5minutes. After this pulse-label period nonradiolabeled UDP-GlcA was thenadded to final concentrations of 900 uM. Samples (100 uL) were takenafter the pulse at 1.5 min (dark circle), and 15 (black square), and 45(black triangle) min after the “chase.” The reactions were terminated bythe addition of SDS to 2% and heating at 95 degrees Celsius for 1 min.The samples were clarified by centrifugation (10,000×g, 5 min) beforeinjection of half of the sample onto a Sephacryl S-500HR gel filtrationcolumn (Pharmacia; 1×50 cm) equilibrated in 0.2 M NaCl, 5 mM Tris, pH 8.

The column was eluted at 0.5 ml/min and radioactivity in the fractions(1 ml) was quantitated by liquid scintillation counting after addingBioSafell cocktail (4.5 ml, Research Products Intl.). The void volumeand the totally included volumes were at elution volumes of 14 ml and35.5 ml, respectively. The peak of blue dextran (average 2×10 6 Da)eluted at 25-27 ml. The recombinant HAS expressed in the eukaryoticyeast cells makes high molecular weight hyaluronic acid in vitro.

Thus it should be apparent that there has been provided in accordancewith the present invention a purified nucleic acid segment having acoding region encoding enzymatically active HAS, methods of producinghyaluronic acid from the seHAS gene, and the use of hyaluronic acidproduced from a HAS encoded by the seHAS gene, that fully satisfies theobjectives and advantages set forth above. Although the invention hasbeen described in conjunction with specific embodiments thereof, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the appended claims.

1. A recombinant host cell comprising at least one expression constructcomprising a hyaluronan synthase gene, wherein the hyaluronan synthasegene encodes an enzymatically active hyaluronan synthase, wherein theenzymatically active hyaluronan synthase includes at least two aminoacid motifs selected from the group consisting of: (i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_(3—7) (SEQ ID NO:11)(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE; (ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (SEQ ID NO: 12)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R); (iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T) (SEQ ID NO:13) XGDDR(H/R/C)LTN;and (iv) (E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N) (SEQ ID NO: 14)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; andwherein the at least two amino acid motifs are found in the relativeorder of (i), (ii), (iii), and (iv) from an amino terminus to a carboxylterminus of the recombinantly produced, enzymatically active hyaluronansynthase.
 2. The recombinant host cell of claim 1, wherein therecombinant host cell produces HA.
 3. The recombinant host cell of claim1, wherein the at least one expression construct further comprises atleast one gene encoding an enzyme for synthesis of a hyaluronic acidsugar precursor.
 4. The recombinant host cell of claim 3, wherein the atleast one gene encoding an enzyme for synthesis of a hyaluronic acidsugar precursor is selected from the group consisting of apyrophosphorylase, a transferase, a mutase, a dehydrogenase, and anepimerase capable of producing UDP-GlcNAc or UDP-GlcUA.
 5. Therecombinant host cell of claim 1, further comprising a second at leastone expression construct having at least one gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 6. The recombinant hostcell of claim 1, wherein the recombinant host cell further comprises atleast one integrated or plasmid-borne gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 7. The recombinant hostcell of claim 1, wherein hyaluronic acid sugar precursors are expressedin the recombinant host cell by endogenous genes thereof.
 8. Therecombinant host cell of claim 1, wherein the hyaluronan synthase geneis isolated from a prokaryotic source.
 9. A recombinant host cellcomprising at least one expression construct comprising a hyaluronansynthase gene, wherein the hyaluronan synthase gene encodes anenzymatically active hyaluronan synthase, wherein the enzymaticallyactive hyaluronan synthase includes at least three amino acid motifsselected from the group consisting of: (i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_(3—7) (SEQ ID NO:11)(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE; (ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (SEQ ID NO: 12)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R); (iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T) (SEQ ID NO:13) XGDDR(H/R/C)LTN;and (iv) (E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N) (SEQ ID NO: 14)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; andwherein the at least three amino acid motifs are found in the relativeorder of (i), (ii), (iii), and (iv) from an amino terminus to a carboxylterminus of the recombinantly produced, enzymatically active hyaluronansynthase.
 10. The recombinant host cell of claim 9, wherein therecombinant host cell produces HA.
 11. The recombinant host cell ofclaim 9, wherein the at least one expression construct further comprisesat least one gene encoding an enzyme for synthesis of a hyaluronic acidsugar precursor.
 12. The recombinant host cell of claim 11, wherein theat least one gene encoding an enzyme for synthesis of a hyaluronic acidsugar precursor is selected from the group consisting of apyrophosphorylase, a transferase, a mutase, a dehydrogenase, and anepimerase capable of producing UDP-GlcNAc or UDP-GlcUA.
 13. Therecombinant host cell of claim 9, further comprising a second at leastone expression construct having at least one gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 14. The recombinant hostcell of claim 9, wherein the recombinant host cell further comprises atleast one integrated or plasmid-borne gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 15. The recombinant hostcell of claim 9, wherein hyaluronic acid sugar precursors are expressedin the recombinant host cell by endogenous genes thereof.
 16. Therecombinant host cell of claim 9, wherein the hyaluronan synthase geneis isolated from a prokaryotic source.
 17. A recombinant host cellcomprising at least one expression construct comprising a hyaluronansynthase gene, wherein the hyaluronan synthase gene encodes anenzymatically active hyaluronan synthase, wherein the enzymaticallyactive hyaluronan synthase includes the amino acid motifs: (i)GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X)_(3—7) (SEQ ID NO:11)(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE; (ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (SEQ ID NO: 12)(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)(I/V/C)(S/G)GPL(G/S)XY(K/R); (iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T) (SEQ ID NO:13) XGDDR(H/R/C)LTN;and (iv) (E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N) (SEQ ID NO: 14)RW(S/T/N)KS(Y/W/F)(F/C)RE,

with X being selected from the group consisting of any amino acid; andwherein the amino acid motifs are found in the relative order of (i),(ii), (iii), and (iv) from an amino terminus to a carboxyl terminus ofthe recombinantly produced, enzymatically active hyaluronan synthase.18. The recombinant host cell of claim 17, wherein the recombinant hostcell produces HA.
 19. The recombinant host cell of claim 17, wherein theat least one expression construct further comprises at least one geneencoding an enzyme for synthesis of a hyaluronic acid sugar precursor.20. The recombinant host cell of claim 19, wherein the at least one geneencoding an enzyme for synthesis of a hyaluronic acid sugar precursor isselected from the group consisting of a pyrophosphorylase, atransferase, a mutase, a dehydrogenase, and an epimerase capable ofproducing UDP-GlcNAc or UDP-GlcUA.
 21. The recombinant host cell ofclaim 17, further comprising a second at least one expression constructhaving at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 22. The recombinant host cell of claim17, wherein the recombinant host cell further comprises at least oneintegrated or plasmid-borne gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 23. The recombinant host cell of claim17, wherein hyaluronic acid sugar precursors are expressed in therecombinant host cell by endogenous genes thereof.
 24. The recombinanthost cell of claim 17, wherein the hyaluronan synthase gene is isolatedfrom a prokaryotic source.
 25. A recombinant host cell comprising atleast one expression construct comprising a hyaluronan synthase gene,wherein the hyaluronan synthase gene encodes an enzymatically activehyaluronan synthase, wherein the enzymatically active hyaluronansynthase includes at least two amino acid motifs selected from the groupconsisting of: (i) GKR(X)₆F(X)₁₁₋₁₅DSDT(X)₈E; (SEQ ID NO:15) (ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y; (SEQ ID NO:16) (iii)QXFXG(X)₅GDDRXLTN; (SEQ ID NO:17) and (iv) P(X)₈QQXRWXKS(X)₂RE, (SEQ IDNO:18)

with X being selected from the group consisting of any amino acid; andwherein the at least two amino acid motifs are found in the relativeorder of (i), (ii), (iii), and (iv) from an amino terminus to a carboxylterminus of the recombinantly produced, enzymatically active hyaluronansynthase.
 26. The recombinant host cell of claim 25, wherein therecombinant host cell produces HA.
 27. The recombinant host cell ofclaim 25, wherein the at least one expression construct furthercomprises at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 28. The recombinant host cell of claim27, wherein the at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor is selected from the group consisting ofa pyrophosphorylase, a transferase, a mutase, a dehydrogenase, and anepimerase capable of producing UDP-GlcNAc or UDP-GlcUA.
 29. Therecombinant host cell of claim 25, further comprising a second at leastone expression construct having at least one gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 30. The recombinant hostcell of claim 25, wherein the recombinant host cell further comprises atleast one integrated or plasmid-borne gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 31. The recombinant hostcell of claim 25, wherein hyaluronic acid sugar precursors are expressedin the recombinant host cell by endogenous genes thereof.
 32. Therecombinant host cell of claim 25, wherein the hyaluronan synthase geneis isolated from a prokaryotic source.
 33. A recombinant host cellcomprising at least one expression construct comprising a hyaluronansynthase gene, wherein the hyaluronan synthase gene encodes anenzymatically active hyaluronan synthase, wherein the enzymaticallyactive hyaluronan synthase includes at least three amino acid motifsselected from the group consisting of: (i) GKR(X)₆F(X)₁₁₋₁₅DSDT(X)₈E;(SEQ ID NO:15) (ii) RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y; (SEQ ID NO:16)(iii) QXFXG(X)₅GDDRXLTN; (SEQ ID NO:17) and (iv) P(X)₈QQXRWXKS(X)₂RE,(SEQ ID NO:18)

with X being selected from the group consisting of any amino acid; andwherein the at least three amino acid motifs are found in the relativeorder of (i), (ii), (iii), and (iv) from an amino terminus to a carboxylterminus of the recombinantly produced, enzymatically active hyaluronansynthase.
 34. The recombinant host cell of claim 33, wherein therecombinant host cell produces HA.
 35. The recombinant host cell ofclaim 33, wherein the at least one expression construct furthercomprises at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 36. The recombinant host cell of claim35, wherein the at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor is selected from the group consisting ofa pyrophosphorylase, a transferase, a mutase, a dehydrogenase, and anepimerase capable of producing UDP-GlcNAc or UDP-GlcUA.
 37. Therecombinant host cell of claim 33, further comprising a second at leastone expression construct having at least one gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 38. The recombinant hostcell of claim 33, wherein the recombinant host cell further comprises atleast one integrated or plasmid-borne gene encoding an enzyme forsynthesis of a hyaluronic acid sugar precursor.
 39. The recombinant hostcell of claim 33, wherein hyaluronic acid sugar precursors are expressedin the recombinant host cell by endogenous genes thereof.
 40. Therecombinant host cell of claim 33, wherein the hyaluronan synthase geneis isolated from a prokaryotic source.
 41. A recombinant host cellcomprising at least one expression construct comprising a hyaluronansynthase gene, wherein the hyaluronan synthase gene encodes anenzymatically active hyaluronan synthase, wherein the enzymaticallyactive hyaluronan synthase includes the amino acid motifs: (i)GKR(X)₆F(X)₁₁₋₁₅DSDT(X)₈E; (SEQ ID NO:15) (ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(X)₂Y; (SEQ ID NO:16) (iii)QXFXG(X)₅GDDRXLTN; (SEQ ID NO:17) and (iv) P(X)₈QQXRWXKS(X)₂RE, (SEQ IDNO:18)

with X being selected from the group consisting of any amino acid; andwherein the amino acid motifs are found in the relative order of (i),(ii), (iii), and (iv) from an amino terminus to a carboxyl terminus ofthe recombinantly produced, enzymatically active hyaluronan synthase.42. The recombinant host cell of claim 41, wherein the recombinant hostcell produces HA.
 43. The recombinant host cell of claim 41, wherein theat least one expression construct further comprises at least one geneencoding an enzyme for synthesis of a hyaluronic acid sugar precursor.44. The recombinant host cell of claim 43, wherein the at least one geneencoding an enzyme for synthesis of a hyaluronic acid sugar precursor isselected from the group consisting of a pyrophosphorylase, atransferase, a mutase, a dehydrogenase, and an epimerase capable ofproducing UDP-GlcNAc or UDP-GlcUA.
 45. The recombinant host cell ofclaim 41, further comprising a second at least one expression constructhaving at least one gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 46. The recombinant host cell of claim41, wherein the recombinant host cell further comprises at least oneintegrated or plasmid-borne gene encoding an enzyme for synthesis of ahyaluronic acid sugar precursor.
 47. The recombinant host cell of claim41, wherein hyaluronic acid sugar precursors are expressed in therecombinant host cell by endogenous genes thereof.
 48. The recombinanthost cell of claim 41, wherein the hyaluronan synthase gene is isolatedfrom a prokaryotic source.